Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review

Journal Pre-proof Biotechnological, biomedical, and agronomical applications of plant protease inhibitors with high stability: A systematic review ´ Juliana Cotabarren, Daniela Lufrano, Monica Graciela Parisi, Walter ´ David Obregon

PII:

S0168-9452(19)31571-7

DOI:

https://doi.org/10.1016/j.plantsci.2019.110398

Reference:

PSL 110398

To appear in:

Plant Science

Received Date:

30 August 2019

Revised Date:

29 October 2019

Accepted Date:

30 December 2019

Please cite this article as: { doi: https://doi.org/ This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

BIOTECHNOLOGICAL, BIOMEDICAL, AND AGRONOMICAL APPLICATIONS OF PLANT PROTEASE INHIBITORS WITH HIGH STABILITY: A SYSTEMATIC REVIEW

Juliana Cotabarren1,*, Daniela Lufrano1, Mónica Graciela Parisi2, and Walter David Obregón1,* 1

of

Centro de Investigación de Proteínas Vegetales (CIProVe-CICPBAUNLP), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 y 115 S/N, B1900AVW, La Plata, Argentina. 2 Departamento de Ciencias Básicas, Universidad Nacional de Luján, Ruta 5 y Avenida Constitución, Luján, 6700 Buenos Aires, Argentina.

ro

E-mail addresses: [email protected] (J. Cotabarren), [email protected] (D. Lufrano), [email protected] (M.G. Parisi), [email protected] (W. D. Obregón).

-p

*Corresponding authors.

ur na

lP

re

Graphical abstract

Jo

Highlights ● Plant PPIs are small molecules with remarkable physical and chemical stability.

● PPIs have a number of applications in biomedicine, biotechnology, food industry and diagnosis.

1

● PPIs belong to the natural compounds with physicochemical stability and biologic activities.

lP

re

-p

ro

of

Abstract Protease inhibitors (PIs) are regulatory proteins found in numerous animal tissues and fluids, plants, and microorganisms that reduce and inhibit the exacerbated and uncontrolled activity of the target proteases. Specific PIs are also effective tools for inactivating proteases involved in human diseases like arthritis, pancreatitis, hepatitis, cancer, AIDS, thrombosis, emphysema, hypertension, and muscular dystrophy among others. Plant PIs—small peptides with a high content of cystine residues in disulfide bridges— possess a remarkable resistance to heat treatment and a high stability against shifts in pH, denaturing agents, ionic strength, and proteolysis. In recent years, novel biologic activities have been reported for plant PIs, including antimicrobial, anticoagulant, antioxidant action plus inhibition of tumor-cell growth; thus pointing to possible applications in medicine, agriculture, and biotechnology. In this review, we provide a comparative overview of plantPIs classifying them in four groups according of their thermal and pH stability (high stability and hyperstable -to temperature and to pHs-, respectively), then emphasizing the relevance of the physicochemical characteristics of these proteins for potential biotechnological and industrial applications. Finally, we analyze the biologic activities of the stable protease inhibitors previously characterized that are the most relevant to potential applications in biomedicine, the food industry, and agriculture.

Jo

ur na

Key words Proteases; Plant Protease inhibitors; Physicochemical stability; Bioactive proteins and peptides

2

Jo

ur na

lP

re

-p

ro

of

Contents 1. Introduction 1.1 Natural products of vegetable origin 1.2 Protease inhibitors 1.3 Plant protein protease inhibitors (PPIs) 1.4 Characterization and potential applications of plant PPIs 2. Applications 2.1 Biomedical applications 2.2 Agricultural applications 2.3 Applications to the food industry 2.4 Significance of the search for stable molecules and their role in the applications discussed thus far 3. Materials and methods 4. Physicochemical stability of plant PPIs 4.1 Thermostable plant PPIs 4.1.1 Plant PPIs with high thermostability 4.1.2 Plant PPIs hyperstable to temperatures 4.2 pH stability of plant PPIs 4.2.1 Plant PPIs with high stability to extreme pHs 4.2.2 Plant PPIs hyperstable to extreme pHs 5. PPIs with physicochemical stability and in-vitro and/or in-vivo biologic activity 6. Concluding remarks

3

Jo

ur na

lP

re

-p

ro

of

1. Introduction 1.1 Natural products of vegetable origin Plants produce natural compounds that act as a major source of bioactive molecules with a wide range of biologic targets [1–3]. Ever since ancient times, nature has provided many bioactive extracts that are rich in molecules for treating diseases, thus being attractive sources of novel compounds for potential drug discovery. Between the first reports on the use of natural extracts with medicinal purposes were the Ebers Papyrus (Egypt 1500 BCE), a record of more than 700 natural extracts with medicinal properties; the Chinese Materia Medica (1100 BCE), containing 52 prescriptions; and the Ayurvedic (India, 1000 BCE) with descriptions of more than 300 natural medicinal extracts. In recent years, the search for natural compounds with medicinal application is increasing because the practice of using synthetic drugs has come under discussion, owing to the high costs of treatment, toxicity from side effects, and reduced efficacy; thus producing the need to complement treatment with natural medications [4]. The mechanisms of antibiotic resistance were reported before the use of antibiotics. Bacteria quickly gain advantageous mutations because the rapidity of bacterial growth enables even the rarest of mutations to become selected. Antibiotics are never effective over the long term, thus spurring a growing interest in the search for novel natural compounds with antibacterial and antifungal activities for application in the biomedical, biotechnological and food industries [5,6]. Current concerns within the fields of industry, agronomy, and food technology are the considerable annual costs on an international scale resulting from losses in crops of agroeconomic value along with a concurrent damage to the environment. These losses can occur because of the attack of pests or through phytopathogenicity from the very agrochemicals directed at preventing and controlling unwanted organisms, which detrimental phytotoxicity encourages the search for bioactive molecules of natural origin. Antimicrobial molecules provide the first line of defense against pathogenic microbes in both plants and animals: indeed, several antimicrobial molecules from plants that inhibit the growth of major agronomic pathogens have been isolated from various plant sources [7–10]. In addition, the growing demand for food worldwide is worrisome from the standpoint of the effective conservation of food, a consideration that once again points to the use of natural products as ecofriendly alternatives to synthetic preservatives. As foodborne pathogenic microorganisms constitute a direct risk to human health, recent years have seen a growing consumer demand for natural products as biopreservatives to replace chemical compounds. Natural antimicrobial molecules could be used to control foodborne pathogens in addition to preserving food [11]. Among these natural compounds, certain ones are different products of the primary and secondary metabolism of plants—e. g., proteins, lipids,

4

carbohydrates, terpenes, polyphenols, and carotenoids. As to proteic molecules, several proteins and peptides with potential biotechnologic and biomedical applications have been isolated and characterized—e. g., chitinases, β-1,3-glucanases, thaumatin-like proteins, endoproteases, peroxidases, ribonuclease-like proteins, γ-thionin and plant defensins, oxalate oxidases, oxalate-oxidase–like proteins, proteases, and protease inhibitors [12,13]. The development of natural products for the prevention and treatment of diseases continues to attract worldwide attention.

Jo

ur na

lP

re

-p

ro

of

1.2 Protease inhibitors Among the natural compounds, protease inhibitors (PIs) are regulatory molecules found in numerous animal tissues and fluids, plants, and microorganisms that control the activity of their target proteases; in some instances blocking their exacerbated and uncontrolled activity [14,15]. The main physiologic function of endogenous PIs is the prevention of unwanted proteolysis and, therefore, in most normal physiologic processes, as well as in pathologic circumstances. That involvement in the regulation of proteolytic activity includes the activation of coenzymes and the release of biologically active polypeptides [16,17]. The presence of PIs in mammalian plasma suggests participation of those enzymes in the regulation of blood coagulation and other proteolytic cascades, such as the activation of complement. The action of enzyme inhibitors in drug research has become a fundamental tool in the pharmaceutical industry but also in the diagnosis or therapeutics of cardiovascular pathologies, cancer, or Alzheimer's disease [13]. Proteolysis is central to many vital biologic processes—such as immunity, blood clotting, cell-cycle regulation, and tissue morphogenesis [18,19]. An external signal can be rapidly amplified through the activation of a single protease that then regulates multiple downstream pathways [20]. Among protease inhibitors, those of a protein nature (PPIs) are small molecules ranging from 15 to 60 amino acids with a high content of cystine residues in the form of disulfide bridges that confer resistance to heat treatment, extreme pHs or ionic strengths, and proteolysis [21–26]. At the present time, PPIs have a number of applications in biomedicine, biotechnology, the food industry, and diagnosis. Most of the pharmaceutical as well as nutraceutical compounds that are marketed are enzyme inhibitors and as such they exhibit a specific action inside cells, bacteria, viruses, animals, plants, and the human body. In whole organisms, these molecules represent an efficient way to control the activity of endogenous proteases, which enzymes need to be balanced in a normal state to effect a controlled proteolysis. Apoptosis, blood clotting, and cellsignalling cascades are some of the processes in which proteases cleave proteins or proenzymes. Since a precise regulation of proteolytic activity is essential for human physiology, many proteases have become principal

5

lP

re

-p

ro

of

biomedical targets [27]. In the example of plant PPIs, a wealth of increasing evidence has demonstrated that those molecules have both preventive and therapeutic effects on several common cancers, as well as on multiple sclerosis, inflammatory processes, and a number of other diseases [28–31]. Studies over recent decades have revealed the positive contribution of PPIs to human health in an increasing fashion. In the food industry, a clear pattern of marketing natural alternatives for food conservation has occurred, resulting from the potential negative effect of synthetic preservatives on consumer health [32]. Another negative effect is the increase in microorganisms manifesting antibiotic resistances and thus being more tolerant to food processing and conservation methods. For this reason, the use of natural antimicrobial compounds as an alternative form of food preservation has been gaining special interest in food science and technology. Consistent with this pattern, we propose that certain PPIs could be explored for their use as natural preservatives of packaged food because of their antimicrobial properties [33–35], especially since they are novel molecules, having never been exposed to microorganisms and therefore, in principle, would encounter no initial microbial resistance. The possibility of using PPIs as preservatives also represents an added value because of their protease-inhibitory capability, as exemplified by the inhibition of proteases in the fish food industry [36–39]. In addition, the antioxidant properties of certain PPIs would add an additional benefit in their use for long-term preservation, by reducing the oxidation in foods [40,41].

Jo

ur na

1.3 Plant protein protease inhibitors (PPIs) In plants, PIs have also been extensively studied owing to their physiologic role in the regulation of endogenous proteases, storage, mechanisms of defense against infection by pathogens, and potential function as antifeedant compounds—as such, protecting plants against herbivorous insects by inhibiting digestive proteases [42,43]. In seeds, tubers and other plant-storage tissues, PPIs represent about 10% of the total protein content, providing the sources of carbon, nitrogen, and sulfur required during germination [44], but the incidence of these proteins in the aerial part of the plants, as a result of several stimuli, has also been extensively documented [45]. The expression of these inhibitors varies according to the maturation stage, tissue location, and time of harvest and storage as well as to the plant variety, with the possible coexistence of different classes of inhibitors and isoforms in a single tissue or organ [46]. High levels of PPIs are often found in plants belonging to the Solanaceae, the Leguminosae (Fabaceae), and the Gramineae (Poaceae) families [47–49]. These proteins, as those from non-plant sources, can be classified according to the catalytic site of the target enzyme. Accordingly, inhibitors against serine, cysteine, aspartic, and metalloproteases have been identified,

6

Jo

ur na

lP

re

-p

ro

of

although plant PPIs against threonine or glutamate proteases have not been reported [26]. Most of the plant PPIs are serine-protease inhibitors [50–52] and as such are classified into more than 20 families. Plant serine-protease inhibitors are widely distributed within the plant kingdom and as such occur in many plant species [52,53], though most have been isolated from the Solanaceae, Fabaceae, Euphorbiaceae, Poaceae, and Cucurbitaceae families. Serine-protease inhibitors are classified in the following groups: Kunitz, Bowman-Birk; Potato I and Potato II; and the superfamily of pumpkin, zucchini, and cereals. New classification systems include groups such as 2Salbumin, Group I and Group II among others. In this review we refer to the last of these inhibitors as other types of serine-protease inhibitors because the majority of the physicochemically stable inhibitors belong to the Kunitz and Bowman-Birk families. Plant PPIs of Kunitz and Bowman-Birk types are often found in members of the leguminous family. These inhibitors are classified on the basis of their cysteine-residue content and the number protein-binding sites. Of the two, the Kunitz-type inhibitors are proteins that usually exhibit a molecular mass of 18–24 kDa, with one or two polypeptide chains and 4 cystine residues forming 2 disulfide bridges, and with a single proteinbinding site [54]. In contrast, the Bowman-Birk inhibitors are small proteins (molecular weight ca. 4–8 kDa) with 14 cystine residues forming seven intrachain disulfide bonds or interstrand disulfide bridges, and with two protein-binding sites [55]. Chiche and colleagues [56] introduced for the first time the pumpkin inhibitor, a now well established family of highly potent serine-protease inhibitors isolated from the Cucurbitaceae family. These pumpkin inhibitors were among the first plant PPIs discovered with the typical so-called crease of Knottin [57]—a protein structural motif containing three disulfide bridges—shared by numerous peptides extracted from plants, animals, and fungi. The second class of more extensively studied inhibitors in plants [58] is the cysteine proteases. Plant cystatins or phytocystatins are classified in 3 subfamilies: Group I, comprising members of molecular mass 12–16 kDa with a single cystatin domain; Group II, of molecular mass 23 kDa and with domains in their N- and C-termini that confer the ability to bind to and thus inhibit cysteine proteases of type C13 [59]; and finally Group III, composed of multicystatins that contain two or more cystatin-type domains [60]. Phytocystatins have been identified and characterized from several plants—namely cowpea [61], potato [62], cabbage [63], ambrosia [64], carrot [65], papaya [66], apple [67], avocado [68], castaño [69], and tears of Job [70]. Cystatins have also been isolated from the seeds of a wide range of crop plants; including sunflower, rice, wheat, maize, soybean, and sugarcane [53,69–71]. In addition, a few aspartate and metalloprotease inhibitors have also been reported [72]. Aspartyl-protease inhibitors—a less extensively studied

7

-p

ro

of

class of peptide inhibitor—have been found in sunflower flowers, barley, thistle (Cynara cardunculus), and potato tubers [13,73–76]. The cathepsin-D inhibitor—an aspartic-protease inhibitor found in potato tubers and containing a considerable amino-acid–sequence homology to the soyatrypsin inhibitor—is a 27-kDa protein that inhibits cathepsin D and the serine proteases trypsin and chymotrypsin, though this plant PPI does not inhibit the aspartic proteases pepsin, cathepsin E, and renin [73]. Metalloprotease inhibitors in plants are scarce, with only five having been isolated to date and all five belonging to the Solanaceae family [25,77– 80]. This group of inhibitors is represented by the metallocarboxypeptidaseinhibitor family present in tomato and potato plants [81,82], of a characteristic molecular mass around 4 kDa and with the latter being the more prevalent and extensively characterized inhibitor. This family of inhibitors typically contains 3 intramolecular disulphide bonds. This structural feature gives a high ratio of disulphide bridges to total number of amino acids that constitute the protein, which turns the molecules into compact and unalterable structures, representing one of the most stable proteins in nature.

Jo

ur na

lP

re

1.4 Characterization and potential applications of plant PPIs PPIs are widely found in crop plants, and though displaying a particular abundance in legumes, are also present in cereals and tubers, where they form part of the plant’s defense against pest attack. That defensive role of PPIs is based on their inhibition of the digestive enzymes of insects and of other pathogenic proteases involved in certain vital processes, either causing a critical shortage of essential amino acids [83,84] or interfering with essential biochemical or physiologic processes [85]. In particular, high levels of PPIs are associated with plant resistance against insects and microbes [12,86]. Although this latter function has been well documented, the mechanism for plant protection per se still remains unclear. That many plant PPIs act as defensive compounds against pests has been demonstrated in direct trials or indirectly by expression in transgenic crop plants [42,87–96]. All of the above characteristics are why PPIs are being incorporated, into the agricultural industry within comprehensive pestcontrol programs. Previous reports have indicated that certain plant PPIs possess special structural characteristics that are responsible for conferring on those peptides a remarkable physical and chemical stability, which properties have generated a special interest in the search and characterization of those molecules. In nature, the number of molecules with physicochemical stabilities such as resistance to high temperatures, extreme pH values, and/or high salinity, among others, is scarce. These characteristics are usually present in several plant PPIs with the so-called cystine knot or crease of Knottin (cf. Section 1.3) that exhibit compact and stable structures joined by

8

ro

of

numerous disulfide bridges relative to the total number of constituent amino acids, with this classification pertaining to many plant PPIs [97]. These features—which constitute special promising qualities in the PPIs in addition to their multiple biologic activities—have occasioned a special interest in the scientific community in undertaking an exhaustive investigation with the main objective of enhancing knowledge regarding additional potential applications of PPIs to those uses already reported. For this reason, the study of plant PPIs opens new paths in scientific research because of the potential use of those proteins in biomedicine, biotechnology, agronomics, and food science, especially in view of the limited number of molecules of natural origin that are already used in those fields. Finally, the purpose of this review is to provide an overview of all the plant-derived PPIs that exhibit high thermal and pH stability, so as to underscore the relevance of this physicochemical stability to the potential application of those compounds in biomedicine, agriculture, and the food industry.

Jo

ur na

lP

re

-p

2. Applications 2.1 Biomedical applications The use of natural products for the prevention or treatment of human diseases continues to be an area of intense research. Approximately 3 billion people—almost half of the world's population—are at a risk of contracting infectious diseases [98]. That danger is greater under poor living conditions, where treatments are inadequate or inaccessible, and in those geographical regions of warm climate and high humidity; with the latter constituting suitable conditions for the development of diseases caused by different types of pathogenic microorganisms. This situation had previously prevailed principally in underdeveloped countries, but now under the influence of the global climate change, has also obtained in regions not previously under risk, but currently exhibiting an increased incidence of several diseases transmitted by warm-climate vectors (i. e., malaria, dengue). An estimation has been made that, between 2030 and 2050, the present climate change could lead to another 250,000 annual deaths caused by malaria, diarrhea, extreme heat, and malnutrition [99]. In addition, the increase in intra- and intercontinental migrations of pathogens also contributes to the arrival of new and emerging infections (i. e., tuberculosis and malaria). New alternative therapies such as homeopathy and ayurvedic medicine are emerging in recent years, on a smaller scale, to provide solutions to diseases of the elderly and and illnesses that derive from the current living conditions (i. e., stress, junk food, competitiveness). That in the near future more than half of the pharmacotherapeutic arsenal will consist of biopharmaceuticals is well known, which transition will represent a great challenge because of the complexity and difficulty in handling those molecules. More than half of all the Food and Drug Administration approved small-molecule drugs were derived structurally from compounds of natural origin [100,101].

9

Jo

ur na

lP

re

-p

ro

of

Natural biopharmaceuticals emerge as new therapeutic alternatives to conventional products obtained by chemical synthesis. Biopharmaceuticals produced from materials of biologic or biotechnologic origin either are sometimes analogous to human proteins or have a close relationship to those molecules. We need to emphasize this characteristic here in order to differentiate those compounds from other proteins used in therapy but of bovine or porcine origin, over which the human-protein–based or -derived biopharmaceuticals have advantages in terms of efficacy and lower side effects, as in the example of certain insulins. In addition, urban populations in developed countries are also subjected to a growing increase in noninfectious illnesses either of genetic origin or caused by modern life with effects on metabolism and general homeostasis—e. g., diabetes, hypertension, obesity, cancer, and Alzheimer's disease. For those diseases, new therapeutic alternatives based on natural products are also being sought. The use of PPIs in biotechnology and pharmaceuticals [102–105], and the potential application of those proteins as valuable tools in the study of the mechanism of action and structure of enzymes have received much attention within the scientific community. The control of proteolysis has been reported as a pharmacologically efficient tool, through the use of different protease inhibitors to treat infectious and systemic diseases. The potentiality and therapeutic efficacy of PPIs have been exemplified in the treatment of immune, inflammatory, respiratory [106], cardiovascular, and neurodegenerative diseases (such as Alzheimer's disease) [107] and have proved useful in drug design to prevent the spread of pathogens that cause dangerous diseases, such as AIDS [108,109], hepatitis [110,111], cancer [112–114] and malaria [115], among others [116,117]. Accordingly, many references can be found in the literature on the beneficial effects that plant PPIs could have on such pathologies. The versatility observed in these natural molecules is truly remarkable, where multiple biologic activities have been reported in relation to antitumor [23], anticoagulant [118], antihypertensive [119] and antioxidant action [40,120] among others. In addition, PPIs have created great interest in studies of protein-protein interaction with valuable relevance to pharmacotherapeutics [121]. As mentioned above, one of the most serious problems for the biomedical field is the growing appearance of multiresistant bacteria worldwide, which pathogens undermine the efficacy of antibiotics and threaten healthcare systems, generating the present so-called antibioticresistance crisis. This situation has been attributed to the excessive use and incorrect prescription of antibiotics among other causes. In contrast, the availability of novel antibiotics is usually reduced as a result of economic and regulatory obstacles [26]. Therefore, the scientific community is also looking for natural therapeutic alternatives to replace those antibiotics that are becoming obsolete or out-dated. That search is why the potential use of PPIs as new antimicrobial agents is promising, not only as a result of the

10

of

remarkable physicochemical stability of those proteins [21,26,122,123], but also because the number scientific articles that report PPIs with antimicrobial activity is constantly increasing [12,34,124–130]. The finding of natural and highly stable molecules with antimicrobial activity is advantageous for science in general, but also because the combination of these characteristics is of particular interest to the pharmaceutical industry. Several types of selective and specific plant PPIs have been purified, characterized, and evaluated for their potential biologic activity and stability. These findings have demonstrated that plant PPIs are highly stable molecules and potent inhibitors of the growth of bacteria, fungi, and even certain viruses; thus making those proteins excellent candidates for use as leading compounds in the development of new antimicrobial agents for medical applications.

Jo

ur na

lP

re

-p

ro

2.2 Agricultural applications In plant biotechnology, interest has arisen in new molecules that counteract diseases of mammals and also in new biotechnologic weapons that serve to prevent microbial attacks on plants and to eradicate those pathogenic microorganisms along with infectious insects, especially in crops of biotechnologic, agricultural, and/or cultural interest. Plants are subjected to a great number of pests and pathogenic infections, whose actions contribute substantially to an overall reduction in crop yield. Chemical pesticides have been used for several decades in order to diminish the damage caused by the different invasive species. The agricultural industries employ numerous pesticides to combat this problem, but those compounds are fraught with serious drawbacks because of a lack of specificity, the development of resistance after prolonged use, and a danger to human health resulting from residual toxicity. For this reason, the inadequate use of many of those agrochemicals in combination with the possibility of resistance acquired by various invading organisms has promoted an acute interest in the search for new alternatives to the treatment of those diseases. Thus, biodegradable biologic control agents and/or natural products constitute the most promising alternatives since those recourses are free of pollutant residues and have a reduced incidence in the development of microbial resistance [131]. The presence of pests is prevalent in warm and humid geographical regions, with unwanted pesticide toxicities often occurring in those areas because of an uncontrolled usage of those compounds, whose structures can undergo alterations through microbial action and the climatic conditions within the environment of dispersal. The chemical stability of synthetic pesticides in the environment is inversely related to the efficiency of the natural degradation processes such as microbial metabolism, photochemical degradation, and chemical hydrolysis. The merely partial degradation of pesticides, however, can lead to the formation of metabolites with high

11

Jo

ur na

lP

re

-p

ro

of

environmental impact [132]. Indeed, the persistence of pesticides in an ecologic matrix depends on the efficiency of those forms of natural biologic and chemical degradation, which reactions often can transform those substances into compounds with higher toxicities [133,134]. Natural degradation processes—such as biodegradation, photodegradation, and chemical hydrolysis—are carried out through reactions of oxidationreduction, hydrolysis, and rupture involving intramolecular reorganization. These reactions occur through the enzymatic activity of microorganisms, the irradiation of ultraviolet light, and the pH of the medium [135,136]. A broad knowledge of these physicochemical variables is very useful in identifying the dominant degradation processes within an ecosystem. A large number of publications have concluded that degradation processes are the major determinants in the persistence and toxicity of pesticides within an agricultural environment [137]. Pest control currently employs, in various pesticides, potent neurotoxins because of their rapid-demolition attributes, which agents not only deteriorate the environment, but pose a threat to public health through food and water contamination or simply through accidental exposure. These problems caused by pesticides and their residues have amplified the need for effective and biodegradable pesticides with higher selectivity [138,139]. Alternative strategies have included the investigation of new types of insecticides or the reevaluation and use of traditional botanical agents for pest control. The latter are renewable, nonpersistent in the environment, do not exert a high selection pressure compared to contemporary pest-control strategies (including the Bacillus thuringiensis toxin), and are relatively safe for the natural enemies of pests, nontarget organisms, and humans [140]. Moreover, the growing global demand for food requires an intelligent application of technologies in agricultural practices. One of the crucial elements limiting crop yields is related to the consumption and destruction of cultivars by insect pests. In the area of pest-insect management with food crops, several studies have evaluated the application of a number of compounds to reduce losses in the field. In recent years, the research on application of compounds with potential insecticide activity and based on plant-derived products have gained prominence. Among those candidates for insect-pest control, plant PPIs represent an attractive class of potential biopesticides with a number of significant features ranging from ecologic friendliness in combination with detrimental effects on a wide variety of plant pests and pathogens. In their evolution, many plants have developed physical and molecular strategies that limit the attack of insect pests but allow the attraction of pollinating insects. One of the most common defenses in plants is the rapid synthesis of PPIs [141]. PPIs perform essential functions in plant biologic systems, including the regulation of proteolytic processes and the participation in defense mechanisms against the attack of organisms such as

12

Jo

ur na

lP

re

-p

ro

of

insects [13]. Plant PPIs constitute effective defense tools owing to an antinutritional action against insect phytopathogenic agents through an inactivation of hydrolytic enzymes or a permeabilization of the pathogen's plasma membrane, thus resulting in growth inhibition [14]. Therefore, those PPIs act against insect pests by binding to the active site of the digestive proteases of a number of insects to block proteolytic activity, thus leading to a decreased, if not complete, disruption of dietary-protein digestion. This action reduces the essential amino acids in the insect intestine, which metabolites are required for insect growth and adult survival [83] as well as for the growth and development of the larvae [142]. In addition, PPIs are involved in programmed cell death [143] and other actions related to plant protection against pests and pathogens [144]. Plant PPIs are present in storage tissues such as seeds and tubers, also in the leaves, flowers, and fruits [145,146]; where those peptides are stored and/or operate as endogenous regulators of proteolytic activity [147]. Plant PPIs also inhibit a broad spectrum of activities including a suppression of nematodes pests and an inhibition of the growth of many pathogenic fungi [148]. In addition, those inhibitors have been reported to effect insect development adversely and could also serve as a transgenic resistance factors [149]. Thus, all of these advantages make plant PPIs ideal candidates for biotechnologic applications, especially in the development of transgenic crops resistant to insect pests. While the main digestive proteases in the midintestines of insects are serine proteases with specificities similar to trypsin and chymotrypsin [150], insect proteases have exhibited differences in the interaction with inhibitors from that observed for commercial bovine enzymes. Hence, to achieve an effective pest-control strategy, the selection of different inhibitors that present high stabilities over a range of conditions is essential along with determining the catalytic characteristics of the proteases of the insect mid-intestine as well as the effects of inhibitors on those enzymic activities. Several plant PPIs—from both the wild-type and nonhost relatives— are more effective than the PPIs of the host plants in the management of insect pests, since the digestive enzymes present in the insect organs have not been adapted to those nonhost PPIs [151,152]. For this reason, one of the investigations in the use of plant PPIs is the generation of transgenic plants that express both the guest and the nonhost PPIs of the plants in the control of insect pests of [153–156]. In recent years, several PPIs from plant seeds with insecticide activity have been reported whose mode of action involves a reduction in the hydrolytic processes of the dietary proteins in the intestine of the insect in order to decrease the availability of endogenous amino acids, particularly those essential for larval development [122,157–159]. Finally, we need to emphasize the role of the fungal diseases that cause damage to crops, mainly because of the diversity of the fungi and the difficulty in controlling those pathogens [160]. On a global level, fungal

13

of

diseases have been increasing with the therapy being limited by several conditions such as a lack of complete effectiveness of the drugs used as well as the adverse side-effects that the latter produce in the plants along with the induction of a resistance in the insects. For this reason, among the most fundamental therapeutic targets is the search for new antifungal alternatives from natural components that can exert biocontrol of those insect pests [94,161,162]. Another example to underscore is the production by several known phytopathogenic fungi of extracellular proteases that play an active role in disease development. Moreover, plants synthesize inhibitory polypeptides that can suppress the proteases produced in an attack by phytopathogenic microorganisms [12]. Finally, several proteins and antimicrobial peptides have been isolated from various types of plants that inhibit the growth of agronomically relevant pathogens [94,163].

Jo

ur na

lP

re

-p

ro

2.3 Applications to the food industry In recent years, the food industry has been forced to search for new preservatives that are both natural and stable in order to extend the half-life of food and to preserve the safety of food quality, whether in a natural way or by altering the microflora of the food, because the deterioration and microbial contamination of food has become a worldwide concern [164,165]. Microbes can not only cause foodborne illnesses but also result in significant economic losses during a postharvest period, with that spoilage through microbial activity being estimated at up to 25% of the food produced [166]. Microorganisms can contaminate food in several ways: at the farm through irrigation water, field workers, insects, or exposure to the feces of wild animals; by inadequate postharvest preservation in the transport vehicles, processing equipment, and washing water; or by contamination from other foodstuffs in the processing plant or marketplace [167]. The demand for minimally processed, easy-to-prepare, and ready-toeat fresh food—as well as the globalization of food trade and distribution from a centralized processing plant—poses major challenges for the safety and quality of food [168]. Traditionally, the control of food deterioration during storage and from pathogenic-bacterial contamination was achieved mainly through antibiotics and the application of conservation techniques such as heat treatment, salting, acidification, and drying. The use of synthetic chemicals, however, has become limited because of undesirable medical consequences stemming from an incomplete elimination of pathogens including cancer, acute toxicity, and teratogenicity. The slow degradation periods, furthermore can lead to pollution of the environment [167]. A negative public perception of chemically synthesized antimicrobials in food has recently generated a growing consumer demand for natural products as biopreservatives to replace chemical compounds. The use of

14

Jo

ur na

lP

re

-p

ro

of

preservatives of natural origin would be beneficial through a decrease in the development of resistance to antibiotics by pathogenic microorganisms or by a strengthening of the immune system in humans, while maintaining the ability to improve the quality and useful life of perishable foods [169]. Plant PPIs constitute a potential alternative to chemical antimicrobials as stable and natural additives for food-preservation processes, not only because of their suppression of the growth of pathogenic microorganisms, but also because of their inhibition of proteases. That PPIs are able to prolong the lifes-pan of various food products is well established, as those agents may delay proteolysis by inhibiting the activity of exogenous and endogenous proteases throughout the conservation and processing of food [170]. The use of an adequate amount of plant PPIs is an effective way to prolong the useful life of many types of seafood, such as salted fish products. This property results in the retardation of several deterioration processes—such as protein degradation caused by the action of endogenous and exogenous proteases— through an inhibition by PPIs during food processing and conservation [38,170]. For example, because the presence of proteases linked to myofibrils is crucial for the degradation of the myofibrillar proteins in fish, PPIs were used to block that protein degradation, thus maintaining the structure of the food [171]. Moreover, certain inhibitors of serine proteases with the ability to increase the level of cholecystokinin by inhibiting trypsin can be useful for reducing the consumption of food in humans [172]. In addition, plant PPIs have been found to possess antimicrobial action on various taxa of food pathogens. Therefore, the study of these compounds has focussed on the antibacterial, antifungal, and antiparasitic properties of the proteins. For this reason, many plant PPIs could be used as potential natural preservatives for food storage, in replacement of synthetic products. In such a scenario, the PPI could act as a natural antimicrobial compound during storage and transport, but losing its activity after the food is cooked so as to avoid possible PPI-related digestive disorders, with itself constituting an additional protein source. The aforementioned considerations could lead to significant advances in the food industry and in the well-being of the community, which advantages, in turn demonstrate the significant role of scientific research in the identification and characterization of new plant PPIs. The potential use of plant PPIs as natural food preservatives, coupled with the beneficial biologic activities mentioned above, point to the possibility of incorporating plant PPIs into foodstuffs as functional ingredients. For these reasons, the scientific and biotechnologic community visualizes a promising future in the use of those molecules as potential stable natural additives capable of maintaining and controlling the microbial balance in stored foods, either through supplementation or addition to the food formulations.

15

Jo

ur na

lP

re

-p

ro

of

2.4 Significance of the search for stable molecules and their role in the applications discussed thus far Molecules of natural origin and low molecular weight with physicochemical stability and biologic activity offer the advantage of ease of use in a wide range of applications in the food industry, agricultural biotechnology, or pharmaceutical science. Thus, the search for novel molecules that meet these characteristics has generated particular interest for science and technology, mainly because of the endless advantages in the employment of those compounds, without the need for special requirements such as storage at cold or warm temperatures, special means of stabilization, preservation in buffers, and/or a requirement for a nonoxidative environment, among others. At the present time, more than 450 biopharmaceuticals are being tested for the treatment of about 100 different diseases. Many of those agents are very labile, having little stability against shifts in pH and/or temperature; while others are macromolecules of high molecular weight, which feature hinders their access to regions of fine vascularization and makes their halflife usually quite short. In addition to these characteristics, within the field of pest control, the hostile environment of an insect's mid-intestine and the optimal pH of pathogenic enzymes necessitate a search for physicochemically stable molecules for use as pesticides. The availability of stable natural molecules with the relevant biologic activities fulfills the criteria for industrial use in the form of a profitable, ecologically friendly, and versatile product that is suitable for a wide geographical distribution with respect to commercialization and use in warm, humid areas—and even in those regions without electricity in underdeveloped countries as well as in developing nations with limited resources for environmentally sustainable measures of pest control. Several studies carried out on certain plant PPIs documented inhibitions under extreme conditions of pH and temperature and attributed this stability to the apparent ability of the inhibitor to reversibly denature via a transient state because of the presence of aromatic residues (tyrosine) generally involved in energy transfer and the formation of intramolecular disulfide bonds [146,173–178]. Other plant PPIs exhibited great resistance to high temperatures where that wide range of functional stability was likewise attributed to the presence of intramolecular disulfide bonds [179,180]. The stability of those inhibitors was observed not only with respect to extreme conditions of pH and temperature but also in the face of strong chemical agents such as detergents, reductants, and oxidants [181]. Several plant PPIs have been purified and found to be highly stable and quite active up to 70 ºC [182]. The general results of thermal stability of those proteins over a wide temperature range (10–100 ºC) and comparison with other findings obtained for those plant PPIs enabled the classification of those inhibitors as high-temperature—tolerant proteins. For most

16

lP

re

-p

ro

of

biotechnological applications, highly thermostable proteins are necessity, as that characteristic increases proteolytic efficiency, which property is a key requirement for commercial exploitation [131]. Likewise, a pH stability study indicated that those plant PPIs were functionally stable within different pH ranges, and even under highly acidic and alkaline conditions (pH 2.0–12.0). Studies aimed at characterizing the dependency of the thermal stability of plant PPIs on pH established the optimal conditions for application of those proteins to the biotechnology involved in the development of transgenic crops resistant to insect pests [183]. As mentioned in previous sections, plant PPIs can be used as biopesticides because, owing to the high stability and pH tolerance of those proteins, they can inhibit the highly alkaline proteases found in the intestinal flora of insects, thus deactivating the digestion mechanism of the food material [14]. In addition, an analysis of the structure of proteases and inhibitors would facilitate their genetic engineering into more potent forms against specific pest species [183]. In recent years, studies focussed on the characterization of plant PPIs have demonstrated that those molecules not only exhibited the aforementioned conditions—i. e., low molecular weight, physicochemical stability, and natural origin—but also a wide range of biologic activities. These features have stimulated the investigation of plant PPIs as promising candidates for potential application in biomedicine, agriculture, and/or the food industry. Those specific objectives constitute the rationale for characterizing the physicochemical parameters determining structural stability for the development of more stable forms for use under a wide variety of environmental conditions.

Jo

ur na

3. Materials and methods This review summarizes the data on the physicochemical stability of plant PPIs as well as some of the biologic properties with potential application in biomedicine, agriculture, and the food industry. In an effort to compile all the information on plant PPIs with physicochemical stability that are available in the public domain, a key-word research—i. e., Plant protease inhibitor, thermal stability, pH stability, physicochemical stability, plant serine protease inhibitor, phytocystatins, plant metalloprotease inhibitor, plant aspartyl protease inhibitor—was performed in the following databases: NCBI-PubMed (http://www.ncbi.nlm.nih.gov/pubmed); Science direct (https://www.sciencedirect.com/); Wiley online library (https://onlinelibrary.wiley.com/); Scholar Google (https://scholar.google.com.ar/). Only plant PPIs reported within the last 20 years were considered.

17

We believe that a comprehensive analysis of the latest research on the physicochemical stability and biologic properties of plant PPIs will assist specialists in the fields of natural products ascertain the full potential of those molecules.

-p

ro

of

4. Physicochemical stability of plant PPIs In accordance with the data analyzed, we have classified plant PPIs in four groups (Table 1): 4.1 Thermostable plant PPIs 4.1.1 Plant PPIs with high thermostability 4.1.2 Plant PPIs hyperstable to temperatures 4.2 pH stability of plant PPIs 4.2.1 Plant PPIs with high stability to extreme pH values 4.2.2 Plant PPIs hyperstable to extreme pH values For each of the groups, we will make a brief characterization of the plant PPIs indicating the target protease, plant origin, inhibition constant, and molecular weight as key parameters. We will also emphasize the abundance of physicochemical stable inhibitors according to the mechanistic group to which they belong. Finally, we will summarize the potential biologic activities reported for the relevant inhibitors.

Jo

ur na

lP

re

4.1 Thermostable plant PPIs A total of 57 PPIs has been reported with high temperature stability, one-third of which—i. e., 19 inhibitors (Fig. 1, Panel A)—exhibited a remarkable stability at very high temperatures. These inhibitors maintained their inhibition after incubation for 30 (Group IIA), 60 (Group IIB) and 120 min (Group IIC) at more than 90 ºC. The remaining two-thirds (38 inhibitors) corresponded to highly stable inhibitors that retained at least 2550% (Group IA), 50-75% (Group IB), or more than 75% (Group IC) of their residual inhibition after incubation at temperatures higher than 90 ºC for different lengths of time (5 min, 10 min, 30 min). Within the context of the growing interest in the characterization of new inhibitory molecules of natural origin with high physicochemical stability, we noted that most of the information on plant PPIs with high temperature stabilities have been reported in the last 5 years, with an average of 6 new stable inhibitors per year (Fig. 1, Panel A). The most abundant thermostable plant PPIs also proved to be the serine-protease inhibitors, with a predominance of the Bowman-Birk inhibitors falling within the hyperstable group (Fig. 1, Panel B). 4.1.1 Plant PPIs with high thermostability Inhibitors with high thermal stability can be classified into the following 3 groups based on the activity that they retain at high temperature.

18

lP

re

-p

ro

of

Group IA: Inhibitors that retain 25–50% of the inhibitory activity at 90 ºC The PPIs arbitrarily classified in this group were considered thermally stable by the investigators who characterized them. Within this group are plant PPIs that retain 25–50% of the original inhibitory activity at temperatures over 90 ºC after 5–10 min incubations (Table 2). Notable members among these inhibitors are the Entada acaciifolia trypsin inhibitor [184], Caesalpinia bonduc trypsin inhibitor[185], Archidendron ellipticum trypsin inhibitor [186], Allium sativum papain inhibitor [187], and a Kunitz trypsin inhibitor from Moringa oleifera seeds [38]. Likewise of note within Group IA are inhibitors that were found to be stable at temperatures below 60 °C with a 50% loss of inhibitory activity at 80 °C—namely, the soybean serine-protease inhibitor [188], a trypsin inhibitor isolated from Albizia amara seeds [189], Sorghum- and maizeprotease inhibitors, and trypsin and chymotrypsin inhibitors isolated from Sorghum bicolor and Zea maize, respectively [190]. Another member of this group is PpyTI, a Kunitz trypsin inhibitor isolated from Poincianella pyramidalis seeds. The PpyTI inhibitory activity was not altered significantly at up to 70 °C, but at 80 °C and 90 °C decreased by 30% and 65%, respectively, and at 100 °C was completely abolished [191]. Similar results were found for C11PI, a Bowman-Birk trypsin and chymotrypsin inhibitor isolated from Cajanus cajan seeds [157] and PPI, a Kunitz trypsin inhibitor isolated from Carica papaya seeds [192]. PCI, a carboxipeptidase inhibitor isolated from Solanum tuberosum tubers, retained the total initial inhibitory activity after treatment at 80 °C for 15 min [193], while a chickpea-trypsin inhibitor maintained 30% of its starting degree of inhibition after incubation at 100 °C [194].

Jo

ur na

Group IB: Inhibitors that retain 50–75% of the inhibitory activity at 90 ºC Table 2 contains a list of plant PPIs arbitrarily classified as Group IB, where we grouped together the inhibitors that retained 50–75% of the original inhibitory activity after incubation at 90 ºC for 5–30 min. Within these plant PPIs are BvvTI, a Kunitz trypsin inhibitor isolated from Bauhinia variegata seeds [195] and RflP1, a Kunitz trypsin inhibitor isolated from Rhamnus frangula leaves [196]. In both examples, the trypsin inhibition was stable at up to 50 °C but thereafter decreased gradually to retain about 30% of the original activity when heated at 80 °C for 30 min. The trypsin inhibition of the PPI PRTI, isolated from Putranjiva roxburghii seeds, was retained at up to 70 °C, though above this temperature decreased slightly, being nevertheless almost 90% retained at up to 80 °C. This inhibitory activity, however, fell sharply above 80 °C and was almost 80% lost at 90 °C [177]. Similar results were observed for CgTI, a Kunitz trypsin inhibitor isolated from Cassia grandis seeds [197] and for IETI, another Kunitz trypsin inhibitor isolated from Inga edulis seeds [198].

19

ro

of

Notable within Group IB were inhibitors that retained their initial activity at up to 70 °C; but, when heated at 100 °C, gradually decreased in activity down to about 20% of the original level by 30 min. Examples of these plant PPIs were SAPI, a Bowman-Birk trypsin and chymotrypsin inhibitor isolated from Solanum aculeatissimum fruits [146], and the Kunitz trypsin inhibitor BmPI, isolated from Butea monosperma seeds [139]; ASPI, isolated from A. sativum bulbs [181]; CFTI-1, isolated from Cassia fistula seeds [199]; and PeTI, isolated from Platypodium elegans seeds [200]. Finally, ILTI, a Kunitz trypsin inhibitor isolated from Inga laurina seeds lost about 20% of the initial activity when incubated at 70 °C for 30 min. When heated to 100 °C, a greater decrease in activity occurred down to 40% of the original value [201]. Similar results were obtained for the Senna tora trypsin inhibitor isolated from S. tora seeds [202]; for ClTI, a Kunitz trypsin inhibitor isolated from Cassia leiandra seeds [203]; and for WBCTI, a Kunitz trypsin and chymotrypsin inhibitor isolated from Psophocarpus tetragonolobus seeds [204].

Jo

ur na

lP

re

-p

Group IC: Inhibitors that retain greater than 75% of the initial inhibitory activity at 90 ºC This last group of plant PPIs with especially high thermal stability corresponds to the Group IC, where we grouped together the inhibitors that retained over 75% of the starting inhibitory activity after 5–30 min at 90 °C. In Table 2 we listed the main characteristics of these inhibitors, with the following being typical examples: CSTI is a Kunitz trypsin inhibitor isolated from the pink powderpuff Calliandra selloi Macbride seeds that remained quite stable at increasing temperatures, retaining more than 75% of the original inhibitory activity after incubation at 80 °C for 30 min. At 100 °C, CSTI lost 50% of the initial activity owing to protein denaturation [175]. Similar results were observed for PDTI, a Kunitz trypsin and chymotrypsin inhibitor isolated from Peltophorum dubium seeds [205], and for CpaTI, a trypsin inhibitor isolated from the seeds of the rattlebox plant Crotalaria pallida. This PPI's inhibition was stable at 80 °C and decreased by only 40% at 100 °C after 30 min [180]. Similar results were also reported for BSKTI, a Kunitz trypsin inhibitor isolated from Glycine max cv. Dull Black seeds [206]; for TfgKTI, a Kunitz trypsin and chymotrypsin inhibitor isolated from Trigonella foenum-graecum seeds [207]; for WeCI, a chymotrypsin inhibitor isolated from seeds of the the wild emmer Triticum dicoccoides [208]; and for YBPCI, a carboxypeptidase A inhibitor isolated from Capsicum annuum seeds [25]. De Souza and colleagues [209] reported a Kunitz trypsin inhibitor isolated from Adenanthera pavonina seeds that exhibited stability under different temperatures in the range of 25 °C to 70 °C for 30 min, but whose inhibitory activity started decreasing at 80 °C to finally reach 61% of its original value at 100 °C. PmTKI, a Kunitz trypsin and chymotrypsin

20

inhibitor isolated from Piptadenia moniliformis seeds, also manifested a similar thermostability [210]. Finally, Prasad and coworkers [211] studied BgPI, a Bowman-Birk trypsin inhibitor isolated from Vigna mungo seeds, whose inhibitory activity against trypsin and chymotrypsin was absolutely stable at up to 80 °C, but decreased marginally (by 15%) when incubated at 90 °C and 100 °C for 30 min.

of

4.1.2 Plant PPIs hyperstable to temperatures In this section we included the protease inhibitors that exhibited extraordinary thermal stability when incubated for several minutes—even for hours—at temperatures above 90 °C, making these molecules some the most thermally resistant found in nature [57,97]. What is remarkable is the predominance of serine-protease inhibitors (84.2%) with the Bowman-Birk inhibitors being the most abundant class within this group (Fig. 1, Panel B).

Jo

ur na

lP

re

-p

ro

Group IIA: Inhibitors that retain the initial inhibitory activity after a 30-min incubation at temperatures over 90 ºC Within Group IIA are plant PPIs that maintain the inhibitory activity at temperatures over 90 ºC after 30 min of incubation (Table 3). Notable among these inhibitors were the following: A barley–cysteine-protease inhibitor isolated from Hordeum vulgare seeds was found to be stable at temperatures below 80 °C, but lost only 15% of the initial activity at 100 °C [212]. SSTI, a Kunitz trypsin inhibitor isolated from Sapindus saponaria and tested at temperatures ranging from 37 to 100 ºC, maintained the initial inhibitory activity at up to 60 ºC for 30 min, but above 60 ºC lost some activity, though retaining 88% of the initial level at up to 100 ºC for 30 min [22]. Similar results were reported for CaTI, a Kunitz trypsin and chymotrypsin inhibitor isolated from C. annuum seeds [213] and for PdKI-4, a Kunitz trypsin inhibitor isolated from Pithecellobium dumosum seeds [214]. rRsBBI, a Bowman-Birk trypsin and chymotrypsin inhibitor isolated from Rynchosia subloblata seeds, was stable upon heat treatment from 37 to 100 ºC, with both inhibitions decreasing by less than 10% at 100 °C for 30 min [215]. Similar results were also observed for SFPI, a Bowman-Birk trypsin and chymotrypsin inhibitor isolated from Clitoria fairchildiana seeds [216], and for a trypsin inhibitor isolated from Phaseolus aureus fruits [72]. Finally, Paiva and colleagues [217], investigating CmTI, a BowmanBirk trypsin inhibitor isolated from Cratylia mollis seeds, reported the total retention of the initial inhibitory activity after 30 min of incubation at increasing temperatures, ranging from 20 °C to 100 °C. A similar thermostability was also observed for KBTI, a Kunitz trypsin inhibitor isolated from G. max seeds [218], and for MpBBI, a Bowman-Birk trypsin inhibitor isolated from Maclura pomifera seeds [219].

21

re

-p

ro

of

Group IIB: Inhibitors that retain the initial inhibitory activity after a 60-min incubation at temperatures over 90 ºC Table 3 lists the plant PPIs arbitrarily classified as Group IIB, where the inhibitors are grouped that—quite remarkably—maintain a total inhibitory activity after 60 min of incubation at temperatures above 90 ºC. Notable among these plant PPIs were WSTI-I and WSTI-IV, both BowmanBirk trypsin inhibitors isolated from G. max seeds, whose inhibitory activities were retained even after heating at 90 °C for 60 min [220]; HSTI, a Bowman-Birk trypsin inhibitor isolated from Hyptis suaveolens seeds, whose inhibitory activity was stable over the temperature range 4–95 °C for 60 min [221]; a trypsin inhibitor isolated from Vigna radiata L. R. Wilczek seeds (14 kDa) that exhibited no significant changes in inhibitory activity for up to 50 min at 90 °C, with only a 10% decrease in activity occurring at 90 °C after 60 min [222]; and VuCys1 and VuCys2, two recombinant cysteineprotease inhibitors isolated from Vignia unguicolata leaves. The inhibitory activity of each of these latter cowpea cystatins against papain was only slightly affected after incubation for 10 min at increasing temperatures ranging from 24 to 100 ºC and was not at all reduced at 100 ºC for up to 60 min [24]. Finally, chuPCI, a carboxypeptidase-A inhibitor isolated from Solanum tuberosum subsp. andigenum cv. Churqueña tubers, retained 95% of the initial inhibitory activity even after a 60-min incubation at 100 °C [80].

Jo

ur na

lP

Group IIC: Inhibitors that that retain the initial inhibitory activity after a 120-min incubation at temperatures over 90 ºC In this last category of plant PPIs exhibiting an extreme hyperthermostability are grouped the inhibitors that maintain the initial inhibitory activity after a 120-min incubation at temperatures over 90 °C. These molecules accordingly are extraordinarily heat-resistant, representing the most thermostable peptides characterized in the present research. Table 3 lists the main characteristics of these inhibitors, where the following examples have been cited: RcTI, a trypsin inhibitor isolated from Ricinus communis seed cake, maintained 80% of the initial inhibitory activity even after heating at 100 °C for 2 h, [122]. JcTI-I, a trypsin inhibitor isolated from Jatropha curcas seed cake, exhibited a slight increase in the original inhibitory activity after heating at 90 ºC for up to 20 min and thereafter lost only about 4–5% of the initial value at up to 120 min [123]. LzaBBI, a Bowman-Birk trypsin and chymotrypsin inhibitor isolated from Luetzelburgia auriculata seeds, maintained 90% of the original inhibitory activity after 120 min at 98 °C [26]. Finally, OsTI 2, a trypsin inhibitor isolated from Opuntia streptacantha seeds, manifested an extreme heat resistance even after incubation for 120 min at 90 °C and maintained the initial inhibitory activity after 60 min of incubation at 120 °C under a 1 kg/cm2 pressure [21].

22

ro

of

4.2 pH stability of plant PPIs Of a total of 54 plant PPIs reported with high pH stability, one-third, or 18 inhibitors (Fig. 2, Panel A), exhibited a remarkable stability—given their proteic nature—at very extreme pHs—i. e., after incubation for 30–120 min at pH 2 and 12. The inhibitors that maintained 50–90% of their original inhibitory activities after incubation at pH 3-4 and 10–11 were classified as highly stable to pH, whereas the inhibitors that retained 70–90% of their activity under the same conditions were classified as hyperstable to pH (Fig. 2, Panel B). As with the thermally stable plant PPIs, a growing interest exists in the characterization of new molecules of natural origin with high pH stability. In the last 5 years, plant PPIs with high stability to extreme pH values have been reported, at an average of 5.4 new stable inhibitors per year (Fig. 2, Panel A). The most frequent plant PPIs with pH stability proved to be the serine-protease type, those being with a predominance of Kunitz inhibitors (Fig. 2, Panel B).

Jo

ur na

lP

re

-p

4.2.1 Plant PPIs with high stability to extreme pHs Table 4 lists the plant PPIs with high stability to extreme pHs, many of which proteins were already described in the previous tables. The following examples were notable within these plant PPIs: An M. oleifera trypsin inhibitor was stable over a wide pH range (5–10) with initial inhibitory activity being mainly retained at pH 10 (at an inhibition of 65%). Under highly acidic (pH 3) or alkaline (pH 11) conditions, however, the inhibitory activity abruptly decreased by 20% and 30%, respectively, compared to the value at pH 10 [38]. The garlic (A. sativum) phytocystatin GPC was found to be quite stable for 30 min in the pH range of 6–8, but the inhibitory activity decreased significantly below pH 6 and above pH 8, dropping to about 10% of the starting activity at a pH of 2–4 and about 20% at a pH of 9–10 [187]. A chickpea-trypsin inhibitor retained 70% of the original inhibitory activity when incubated at pH 3–5 for 24 h, whereas at pH 2, 11, and 12 the activity dropped to 5% of the initial value [194]. The inhibitory activities of the plant PPIs OsTI 2 [21] and API [189] remained stable after being incubated at pH 3 and pH 7–8 for 1 h, but upon incubation at pH 9 the activities decreased by 40%. The starting inhibitory activities of the CgTI [197] and BmPI [139] were maintained by over 80% for 1 h between pH 5 and 10 at 37 °C but then declined at those extreme values, while the CFTI-1 [199] and TfgKTI [223] retained a good stability at pHs from 3 to 10 for 30 min, though outside that pH range the stability of those inhibitors was poor (10%) with instablity occurring at extreme pH values. The RflP1 [196], the maize and Sorghum PPIs [190], the soybean PPI [188], and the PPIs SAPI [146] and RcTI [122] all retained ca. 50–80% of the starting inhibitory activities when incubated

23

-p

ro

of

at pH 2–3 or pH 11–12 for 60 min. In Table 4 are also grouped inhibitors that maintained 90% of the initial inhibitory activity over a pH range of 2–10 for 30–60 min—namely, CmTI [217], ILTI [201], SSTI [22], EATI [184], CaTI [213], PPyTI [191], JcTI-I [123], CFPI [216], CITI [203], IETI [198], and PeTI [200]. The trypsin inhibitor from V. radiata L. R. Wilczek seeds was stable over a broad pH range, but exhibited some decrease in activity at low and high pHs, although being generally stable in the neutral range [222]. The inhibitory activity of HSTI was stable at pHs from 3 to 10.7 for 60 min [221]. Incubation of BvvTI at pHs between 4 and 12 for 30 min did not affect subsequent trypsin inhibition [195]. AeTI retained the original inhibitory activity at very alkaline pHs (>12) at which only 34% of its potency was lost after 2 h, with extremes of pH having negligible effects on the inhibition [186]. Plant PPIs such as SPCI, a Kunitz chymotrypsin inhibitor isolated from Schizolobium parahyba seeds [224], CSTI [175], and LzaBBI [26] retained over 80% of the starting inhibitory activity between pH 2 and 11 for 10–30 min. Finally, ASPI [181], YBPCI [25], and the trypsin inhibitor from P. aureus [72] proved to be quite stable within the range pH 2–12, retaining about 70% of the original inhibitory activity after 1 h.

Jo

ur na

lP

re

4.2.2 Plant PPIs hyperstable to extreme pHs Table 5 lists the plant PPIs arbitrarily classified as hyperstable at extreme pHs, retaining more than 70% of the initial inhibitory activity after incubation at pH 2 or 12 for at least 10–120 min, the biochemical characteristics of which proteins are detailed in tables 2 and 3. Notable among these plant PPIs is ECTI, which maintained the total original inhibitory activity after incubation in the pH range 2.0–12.0 for 10 min [225], and PRTI [177], KBTI [218], PmTKI [210], and PdKI-4 [214] plus the S. tora trypsin inhibitor [202], MpBBI [219], VuCys1, VuCys2 [24], and WeCI [208], all of which peptides maintained over 70% of their starting inhibitory activity after incubation for 30 min at pHs from 2 to 12. Likewise, within this group were STI-I and WSTI-IV [220], PDTI [205], CpaTI [180], BgPI [211], C11PI [157], and rRsBBI1 [215] that maintined more than 80% of the initial inhibitory activity for 60 min at extreme pHs. Finally, inhibitors such as PPI [192], CbTI [185], BSKTI [206], and the barley-protease inhibitor [226] retained more than 80% of their original inhibitory activity upon incubation at extreme pHs for more than 120 min. 5. PPIs with physicochemical stability and in vitro and/or in vivo biologic activity In conducting this review, we performed an exhaustive literature search in order to classify plant PPIs according to their stability at extreme

24

pHs and temperatures. In addition, we noted that most of the entries also possessed biologic activities that could be investigated in order to ascertain their potential applicability to biotechnology, biomedicine, agriculture, or the food industry. To that end, we compiled some of the biologic activities reported for several of the plant PPIs that were reviewed—namely, the antimicrobial (both antibacterial and antifungal), the antiviral, the anticoagulant, the antitumoral, and the pesticide activities (Table 6).

ro

of

5.1 Plant PPIs with pesticide activity Plant PPIs with pesticide activity and stability at extreme pHs and temperatures are the most extensive class of inhibitors studied and reported to date. In Table 6 we summarized these inhibitors and classified them according to: 1) inhibition of larval-gut proteases, 2) inhibition of larval development and 3) inhibition of both larval-gut proteases and larval development.

Jo

ur na

lP

re

-p

5.1.1 Inhibitory activity on larval-gut proteases In this group are classified the following plant PPIs that displayed activity towards larval-gut proteases: HSTI, whose inhibitory activity was potent toward all trypsin-like proteases extracted from the gut of the insect Prostephanus truncatus, the larger grain borer, a major pest of maize. This activity was highly specific because, among the proteases from seven different insects, only those from P. truncatus and the tobacco hornworm Manduca sexta were inhibited [221]. CbTI inhibited the gut proteases of Spodoptera litura, the cotton leafworm aka the tobacco cutworm [185]. OsTI 2, inhibited the trypsin-like proteases of the insects P. truncatus; Periplaneta americana, the American cockroach; and the crickets Acheta sp. and Gryllus sp. [21]. SSTI inhibited the larval digestive enzymes extracted from the Anagasta kuehniella, the Mediterranean flour moth aka the mill moth; Corcyra cephalonica, the rice-meal moth; and Diatreae saccharalis, the stem borer. SSTI also inhibited, albeit moderately, the tryptic activity of Anticarsia gemmatallis, the velvetbean caterpiller [22]. Finally, the purified S. tora trypsin inhibitor effectively inhibited total protease and trypsin-like activities of a midgut preparation from the cotton bollworm Helicoverpa armigera [202]. 5.1.2 Inhibition of larval development Among the plant PPIs with biologic activity are the following that inhibit larval growth and development: PDTI, the Kunitz trypsin and chymotrypsin inhibitor isolated from P. dubium seeds and member of Group IC, delayed the development of the flour moth Anagasta (Ephestia) kuehniella [205]. AeTI proved to be an insecticide against the cotton leafworm S. litura [186], whose gut proteases were inhibited by CbTI. Maize and Sorghum PPIs caused larval mortality ranging from 10 to 77% when

25

ur na

lP

re

-p

ro

of

added to the diet of the African cotton leafworm Spodoptera littoralis; with the larval mortality caused by the maize PPI being significantly higher than that of the Sorghum protein, attaining a maximum mortality of 77% when the larvae were fed a diet containing 0.5% (w/w) of the maize PPI [190]. In bioassays performed with the Mediterranean meal moth (Anagasta kuehniella), PpyTI, the Kunitz trypsin inhibitor isolated from Poincianella pyramidalis seeds and a member of Group IA, produced a significant decrease in larval weight and survival, as well as an extension of the larval stage [191]. A purified soybean serine-protease inhibitor markedly reduced the mean larval and pupal weight and caused larval and pupal mortality of S. littoralis [188]. A barley-protease inhibitor significantly increased the mortality and caused a significant reduction in the fecundity of the cowpea seed beetle Callosobruchus maculatus; without, however, having any significant effect on adult longevity and dry weight [226]. CFTI-1, isolated from Cassia fistula seeds and a member of Group IB, produced a concentration-dependent reduction in the mean larval weight, the fertility, and the fecundity of moths plus an extended duration of the life cycle of the cotton bollworm H. armigera [199]. WeCI, the chymotrypsin inhibitor isolated from T. dicoccoides seeds and a member of Group IC, significantly inhibited—in a concentration-dependent fashion—the growth and development, increased the mortality, and decreased the fertility of the beet armyworm Spodoptera exigua [208]. In-vivo assays with CgTI, the Kunitz trypsin inhibitor isolated from C. grandis seeds and a member of Group IB, increased the mortality of the tree termite Nasutitermes corniger [197]. Finally, bioassays with PeTI, isolated from P. elegans seeds and a member of Group IB, on the fall armyworm Spodoptera frugiperda, indicated reductions in larval development and weight gain along with an extension the insect life cycle. Moreover, activities of the digestive enzymes trypsin and chymotrypsin were reduced by feeding the larvae an artificial diet containing 0.2% (w/w) of the PPI [200].

Jo

5.1.3 Inhibition of larval-gut proteases and larval development In the group of inhibitors with pesticide activity, certain ones also exhibited both in-vitro and in-vivo activities: for example, an inhibition of larval-gut proteases as well as a retardation of larval growth and development. CpaTI, the trypsin inhibitor isolated from C. pallida seeds and a member of Group IC, inhibited—to different degrees—the digestive enzymes in the larval guts of S. frugiperda, the cotton leafworm Alabama argillacea, the Indian meal moth Plodia interpunctella, the Mexican cotton boll weevil Anthonomus grandis, and the Mexican bean weevil Zabrotes subfasciatus. This plant PPI also strongly inhibited various trypsin-like enzymes in-vitro and in-vivo of C. maculatus and the mediterranean fruit fly Ceratitis capitata. When CpaTI was added to the artificial diets of both insect larvae, C. maculatus proved to be the more susceptible and C.

26

Jo

ur na

lP

re

-p

ro

of

capitata more resistant to the effects of the protein. The larvae were more affected at lower concentrations; and the action was constant at a dietary concentration of 2–4% (w/w), resulting in a 15% mortality and a 38% decrease in body mass [180]. A chickpea trypsin inhibitor exhibited action against H. armigera both in vivo and in vitro. In a bioassay on the insect, the addition of the plant PPI to the larval diet resulted in a progressive decline in larval weight, growth, and survival; an extension of larval-maturation time; and an inhibition of adult emergence [194]. PmKTI, the Kunitz trypsin and chymotrypsin inhibitor isolated from P. moniliformis seeds and a member of Group IC, reduced markedly the activity of trypsin-like enzymes from A. grandis (90%), the Indianmeal moth Plodia interpuncptella (60%), and C. capitata (70%). Furthermore, an in-vivo bioinsecticidal effect on C. capitata larvae was also demonstrated [210]. In-vitro assays with C11PI, the Bowman-Birk trypsin and chymotrypsin inhibitor isolated from C. cajan seeds and a member of Group IB, demonstrated a significant inhibition—i. e., at a concentration required for 50% inhibition, IC50, of 78 ng—against the midgut trypsin-like proteases of the croton caterpiller Achaea janata. In addition, in-vivo leaf-coating assays demonstrated that C11PI caused a significant mortality, a concomitant reduction in body weight of both larvae and pupae, a prolonged duration of the transition from larva to pupa, and the formation of abnormal larval-pupal and pupal-adult intermediates [157]. Invitro assays with BmPI, the Kunitz trypsin inhibitor isolated from B. monosperma seeds and a member of Group IB, exerted an inhibition of the total gut proteases of H. armigera (IC50 of 2.0 g/ml); and when supplemented in an artificial diet, caused a dose-dependent mortality and reduction in growth and weight, a decline in fertility and fecundity, and an extension of the larval–pupal duration within the insect's life cycle [139]. CFPI exhibited a substantial inhibition of the larval-midgut trypsin of A. kuehniella (76%), the sugarcane borer Diatraea saccharalis (59%), and the tobacco budworm Heliothis virescens (49%). The insecticidal properties of this plant PPI were further analyzed by bioassays and confirmed by a negative impact on A. kuehniella development [216]. WBCTI, the Kunitz trypsin and chymotrypsin inhibitor isolated from seeds of the cottonbollworm P. tetragonolobus and a member of Group IB, inhibited the midgut proteases of H. armigera; and when included in an artificial diet, resulted in a significant growth retardation, a delayed pupa formation, and a higher mortality of larvae [204]. Finally, rRsBBI1 manifested a significant inhibition (IC50 of 70 ng) of the larval-gut trypsin-like proteases (AjGPs) of A. janata. Conversely, this plant PPI exhibited an inhibition (IC 50 of 8 mg) of the larval-gut trypsin-like protease of H. armigera that was only <1% of the activity on the AjGPs. In addition, in-vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on A. janata larval growth and development, which results suggested that these properties of this plant PPI can be further exploited [215].

27

Finally, in Table 6 we reported the inhibitory activity of CITI, the only plant PPI with physicochemical stability plus an insecticidal activity against the yellow-fever mosquito Aedes aegypti. This plant PPI, at a concentration of 4.65 ×10−6 M, reduced the activity of Ae. aegypti midgut proteases by 50% and promoted an acute toxicity in the 3rd-instar larvae in addition to causing a 24-h delay in larval development along with a 44% mortality after ten days of exposure [203].

ur na

lP

re

-p

ro

of

5.2 Plant PPIs with antimicrobial activity Table 6 lists the following plant PPIs with physicochemical stability and antifungal and/or antimicrobial activity: RcTI, the trypsin inhibitor isolated from R. communis seed cake and a member of Group IIC, possesses antifungal activity as evidenced in an inhibition of the spore germination of Colletotrichum gloeosporioides [122]; while IETI, the Kunitz trypsin inhibitor isolated from I. edulis seeds and a member of Group IB, exhibited an antimicrobial activity against Candida ssp., including Candida buinensis and Candida tropicalis triggering a membrane permeability in those yeast, thus decreasing cell viability [198]. JcTI-I, the trypsin inhibitor isolated from J. curcas seed cake and a member of Group IIC, inhibits the growth of the bacteria Salmonella enterica subsp. enterica serovar choleraesuis and Staphylococcus aureus [123]. LzaBBI, the Bowman-Birk trypsin and chymotrypsin inhibitor isolated from L. auriculata seeds and a member of Group IIC, inhibits S. aureus by disrupting the bacterial membrane [26]. Finally, certain inhibitors exhibited both antifungal and antimicrobial activity: API was found to have antifungal activity against Alternaria alternata, Alternaria tenuissima, and Candida albicans as well as antibacterial activity against Pseudomonas aeruginosa and Bacillus subtilis [189], while RflP1, the Kunitz trypsin inhibitor isolated from R. frangula leaves and a member of Group IB, acted as a potent inhibitor of certain commercial bacterial proteases from Aspergillus oryzae, Bacillus sp., and Bacillus licheniformis and also possessed an appreciable antibacterial action against both Gram-positive and -negative bacteria [196].

Jo

5.3 PPIs with antiviral and antitumoral activity Table 6 also lists the following plant PPIs with physicochemical stability that exhibit antiviral and antitumoral activity: BSKTI, the Kunitz trypsin inhibitor isolated from G. max cv. Dull Black seeds and a member of Group IC, possesses potential for being developed into an agent for anticancer therapy in view of the protein's potent antiproliferative activity toward tumor cells and the ability to elicit nitric-oxide production from macrophages. This PPI possesses potent inhibitory activity against HIV-1 reverse transcriptase and hence is also a potential antiretroviral agent. BSKTI resembles the soybean-trypsin inhibitor from G. max cv. Dull Black seeds in the ability to inhibit trypsin and chymotrypsin as well as tumor-cell

28

ro

of

proliferation, but differs in that this PPI can stimulate macrophage nitricoxide production and inhibit HIV-1 reverse transcriptase and a cell-free translation system. BSKTI appears to be more versatile than the soybean trypsin inhibitor in relation to its biologic activity [206]. BvvTI, the Kunitz trypsin inhibitor isolated from B. variegata seeds, possesses an anti-HIV-1 reverse-transcriptase activity and was able to significantly and selectively inhibit the proliferation of the human nasopharyngeal-cancer CNE-1 cells. This action may have been partially mediated by an induction of cytokines and apoptotic bodies [195]. Finally, KBTI, the Kunitz trypsin inhibitor isolated from G. max seeds and a member of Group IIA, inhibited HIV-1 reverse-transcriptase activity with an IC50 value of 0.71 μM and induced the transcription of the mRNAs encoding proinflammatory cytokines such as TNF-α, IL-1β, IL-2, and interferon-γ. This plant PPI also exerted a weak antiproliferative activity toward CNE-2 and HNE-2 nasopharyngeal-cancer cells, MCF-7 human breast-cancer cells, and Hep G2 human-hepatoma cells, but is devoid of mitogenic, ribonuclease, and antifungal activities [218].

ur na

lP

re

-p

5.4 Plant PPIs with anticoagulant activity This group comprises the following PPIs: ECTI, the PPI hyperstable to extreme pHs and a trypsin inhibitor that also affected the blood-clotting parameters—such as the thrombin-clotting time (TT), the prothrombin time (PT), and the activated partial-thromboplastin time (APTT)—when assayed with normal human citrated plasma. The increase in APTT observed indicated that ECTI blocked the contact phase of blood clotting (n = 6; p <0.01), but not the TT and PT, which times were not significantly altered by the PPI [225]. MpBBI, the Bowman-Birk trypsin inhibitor isolated from M. pomifera seeds, also exhibited anticoagulant activity, increasing the APTT but not the TT or the PT, suggesting its potential use in the treatment of bloodcoagulation disorders [227].

Jo

6. Concluding remarks During the most recent decades an increasing interest has developed in the use of natural products for different applications. The reasons for this occurrence are diverse, including a lower toxicity to human and animal health and the broad range of chemical structures and biologic activities found among natural products in combination with a dramatic increase in the number of antibiotic-resistant bacterial strains with the development of novel antibacterial drugs lagging behind [228]. Among the naturally occurring compounds, proteins and peptides are widely explored for various applications, but that class of molecule often has certain drawbacks—e. g., low chemical and/or physical stability, susceptibility to proteolysis and oxidation, and low water-solubility—that have to be overcome by

29

Jo

ur na

lP

re

-p

ro

of

introducing rational modifications, in order to make those agents suitable for applications [229]. In this respect, plant PPIs offer certain advantages, being small molecules with satisfactory solubility in aqueous solutions and resistant to proteolysis. These PIs of vegetable origin not only display several proven invitro and/or in-vivo bioactivities—e. g., pesticidal, antimicrobial, antiviral, antitumoral, anticoagulant—but also, in some instances, exhibit unique stabilities at high temperatures (up to 100 ºC for several minutes, even hours) and at extreme acidities and/or basicities (pH values of 1–2 and 13– 14). Such a physicochemical stability ranks plant PPIs among the most thermally resistant molecules in nature [57,97] and constitutes for those proteins an extra asset over synthetic compounds in numerous potential applications. In biotechnology, these features enable, for example, an inhibition of proteases for the enhancement of yields in processes optimized at extreme temperatures or pHs. As food preservatives, stability at low pHs makes plant PPIs suitable for use in dairy products, while in pharmaceutical research and the development of orally administered drugs, the property of acid tolerance ensures gastric molecular stability. In addition, a high pH and thermal stability may reduce the transportation and storage costs of any product. These characteristics take on special relevance in developing countries that usually face higher trade costs and poorer living conditions [230]. Since many of those countries also have warm climates—e, g., the north of South America, Central America, the Caribbean Islands, Central Africa, the south of Asia, and the north of Oceania—the design of PPI-based biopharmaceuticals and food preservatives that retain their activity at temperatures in the range of 45-60 ºC for long periods of time (6 months to a year) represents the solution to a challenge that the world has to deal with. Despite being a critical issue, in the literature we found very few reports on the combined effects of pH and temperature on plant PPIs—e. g., HSTI [221] and OSTI 2 [21]. Even, for some of the plant PPIs listed the thermal, but not the pH, stability had been tested. In contrast, we found a plethora of conditions (incubation time, temperature, and/or pH) employed in thermal- and pH-stability assays. In order to provide comparable data and obtain broader information on the potentiality of these proteins, a consensus on the conditions for testing the stability of plant PPIs would need to be established. Even though the criteria for those guidelines would be based on the requirements of each type of application; as a starting point, we suggest testing a protocol based on the conditions assayed by Torres-Castillo and colleagues [21], who simultaneously tested the pH and thermal stability of a trypsin inhibitor from O. streptacantha Lemaire seeds by incubating the PPI for 2 h over a range of temperatures from 30 to 120 °C and pHs from 3 to 9. According to the resistance observed in those plant PPIs that we classified as hyperstable to extreme pHs, we propose an extension of the tested pH values to include the range from 1 through 13. The lower limit of that proposed

30

Jo

ur na

lP

re

-p

ro

of

interval includes the gastric pH of normal individuals (from 1 to 4) and the aforementioned use of acidophilic microorganisms in biotechnology [231], while the upper limit not only covers typical duodenal pHs (from 5 to 9; [232]) but also those of the alkaliphiles so widely used in industrial and therapeutic applications [233,234]. With respect to the times to be tested, the longest incubation time most frequently found in literature was 3 h, but we consider that this length of time is an inappropriate choice for those PPIs intended for use as an oral drug, which would require stabilities for as long as 5–6 h in order to traverse the complete digestive tract. As long-term thermal stability is also desirable, accelerated stability tests based on general worldwide protocols (e. g., 40 °C and 75% relative humidity for 1 to 6 h) should be assayed [235]. Data related to the effects of ionic strength, organic solvents, oxidants, and denaturing agents on the stability of plant PPIs are scarce in the literature. Only one of these parameters is reported for 24 of the 57 plant PPIs we reviewed ([192], [184], [189], [201], [177], [195], [181], [203], [196], [200], [205], [175], [206], [211], [210], [22], [213], [216], [215], [21], [26], [207], [222], [123]). While for RcTI [122], CbTI [185], and the barleyprotease inhibitor [226] none of these parameters were studied despite the great potential in industry inherent in proteins with a hyperstability to at least both pH and temperature. Another general lack of information is present with respect to the absence of isoelectric-point (pI) and chargedistribution data in the listings of plant PPIs, parameters which determine the pH range for a formulation based on a PPI that is necessarily soluble. Similarly, simulated gastric digestions, despite being an extremely useful test on potential candidates for the development of oral biopharmaceuticals or food additives, was only assayed for the single plant PPI: YBPCI, the carboxypeptidase A inhibitor isolated from C. annuum seeds and a member of Group IC [25]. As to the inhibitory activity, ca. 20% of the plant PPIs retrieved in the analysis left that extremely relevant information unquantified ([194], [188], [204], [196], [180], [206], [218], [221], [222], [24], [21]). For the remaining 80% of the plant PPIs surveyed, the Ki (inhibition constant) was usually reported, although for PCI [193] and YBPCI [25], the IC50 was given. Even when both parameters, the Ki and the IC50, characterized the potency of the PPIs, the Ki made comparison among different plant PPIs easier and also enables an identification of the type of inhibition—i. e., competitive, noncompetitive, and uncompetitive. The mechanism of inhibition not only gives relevant information about the inhibitor per se but also is a detail required for the conversion of the Ki to the IC50 [236]. In general, the plant PPIs cited in this review have potential application to the generation of genetically modified insect-resistant crops as a result of the pesticide activity described in vitro for the isolated PPIs. This activity has been determined in most examples by assaying larval

31

Jo

ur na

lP

re

-p

ro

of

development ([205], [186], [191], [188], [226], [199], [208], [197], [200], [190])—and to a lesser extent by measuring the inhibition of larval-gut proteases ([221], [185], [21], [22], [202]). In the literature several reports described transgenic plants harboring a transected plant-PPI gene engineered to confer insect-resistance [153–159]. Since, however, genetically modified crops are still a controversial issue in many countries, the external use of plant PPIs, demonstrating physicochemical stability and in-vitro pesticide activity for the biologic control of pests in crops would be a compromise. In the analysis carried out in this review, CpaTI [180], PmTKI [210], C11PI [157], BmPI [139], CFPI [216], WBCTI [204], rRsBBI1 [215], and the chickpea-trypsin inhibitor [194] emerge as promising potential candidates for this application. In addition to the potential applicability inherent in the inhibition exhibited by certain plant PPIs, the protein nature of these molecules enables their adaptation to potential nutraceutical compounds as rich sources of bioavailable amino acids in general and of cysteine in particular. Contrary to the desirable high physicochemical stability discussed so far, the plant PPIs susceptible to degradation at 100 ºC and pHs of 1–4 would be more suitable as nutraceuticals in order to avoid the inhibition of digestive proteases and antinutritional effects. Thus, a simple way to take advantage of this property could be to use plant PPIs with antimicrobial and antioxidant activity as raw food preservatives that finally after a brief cooking (for example by boiling) would become inactivated. Furthermore, these aspects also raise questions about the raw-foods diet, which approach has cited numerous benefits from consuming uncooked foods such as vegetables, fruits, grains, and seeds [237]. Despite the popularity of this kind of a diet, the plethora of plant PPIs in many foods consumed with considerable frequency—such as potato, tomato, eggplant, and sweet pepper—nevertheless leaves a nutritional gap that should be considered. Because certain plant PPIs exhibit gastrointestinal stability and are absorbed in the gut as intact and active proteins [238,239], a quantification of the concentration of those PPIs reaching the portal circulation might be informative with respect to making an estimation of the potentially negative impact on endogenous proteases (or physiologic processes) of a raw diet that may counterbalance the claimed benefits in patients with special conditions. In conclusion, in this review we stressed the need to search for plant PPIs with remarkable physicochemical stabilities and potential biologic activities in order to increase the number of molecules that could act as novel therapeutic agents, food preservatives or biopesticides, among other uses. Our objective was to provide a comparative study of the stability to temperature and extreme pHs of the plant PPIs isolated in the last 20 years. Moreover, we also have described the in-vitro and in-vivo biologic activities

32

reported to date, with an aim at providing available information that can be used by the scientific and industrial communities. Conflicts of interest The authors declare no conflicts of interest.

Jo

ur na

lP

re

-p

ro

of

Acknowledgements This work was supported by Universidad Nacional de Luján (Departamento de Ciencias Básicas Finalidad 3.5) and UAV 2017 Project (Res SPU 5157/17) grants to MGP, PICT-2016-4365, and Universidad Nacional de La Plata (PPID X/014 and PPID X/038) grants to WDO and to DL. J. Cotabarren is a posdoctoral fellow from the Argentine Council of Scientific and Technical Research (CONICET); D. Lufrano is member of the Researcher Career Program of CICPBA and researcher of UNLP; M.G. Parisi is an established researcher of UNLu and W.D. Obregón is member of the Researcher Career Program of the CONICET. Dr. Donald F. Haggerty, a retired academic career investigator and native English speaker, edited the final version of the manuscript.

33

Jo

ur na

lP

re

-p

ro

of

References [1] A.L. Harvey, Natural products in drug discovery, Drug Discov. Today. 13 (2008) 894–901. doi:10.1016/j.drudis.2008.07.004. [2] A.L. Harvey, R. Edrada-Ebel, R.J. Quinn, The reemergence of natural products for drug discovery in the genomics era, Nat. Rev. Drug Discov. 14 (2015) 111. https://doi.org/10.1038/nrd4510. [3] P.A. Clemons, N.E. Bodycombe, H.A. Carrinski, J.A. Wilson, A.F. Shamji, B.K. Wagner, A.N. Koehler, S.L. Schreiber, Small molecules of different origins have distinct distributions of structural complexity that correlate with protein-binding profiles, Proc. Natl. Acad. Sci. 107 (2010) 18787–18792. doi:10.1073/pnas.1012741107. [4] R.A. Otvos, K.B.M. Still, G.W. Somsen, A.B. Smit, J. Kool, Drug Discovery on Natural Products: From Ion Channels to nAChRs, from Nature to Libraries, from Analytics to Assays., SLAS Discov. Adv. Life Sci. R D. 24 (2019) 362–385. doi:10.1177/2472555218822098. [5] G. Cox, G.D. Wright, Intrinsic antibiotic resistance: mechanisms, origins, challenges and solutions., Int. J. Med. Microbiol. 303 (2013) 287–292. doi:10.1016/j.ijmm.2013.02.009. [6] J.M.A. Blair, M.A. Webber, A.J. Baylay, D.O. Ogbolu, L.J. V Piddock, Molecular mechanisms of antibiotic resistance., Nat. Rev. Microbiol. 13 (2015) 42–51. doi:10.1038/nrmicro3380. [7] H.G. Boman, Antibacterial peptides: basic facts and emerging concepts., J. Intern. Med. 254 (2003) 197– 215. [8] P. Bulet, R. Stocklin, L. Menin, Anti-microbial peptides: from invertebrates to vertebrates., Immunol. Rev. 198 (2004) 169–184. [9] L.C. van Loon, M. Rep, C.M.J. Pieterse, Significance of inducible defense-related proteins in infected plants., Annu. Rev. Phytopathol. 44 (2006) 135–162. doi:10.1146/annurev.phyto.44.070505.143425. [10] M.G. Parisi, S. Moreno, G. Fernández, Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities, Plant Physiol. Biochem. 46 34

Jo

ur na

lP

re

-p

ro

of

(2008) 403–413. doi:10.1016/j.plaphy.2007.11.003. [11] T. Hintz, K.K. Matthews, R. Di, The Use of Antibiotics for Food Preservation*, Biomed Res. Int. 2015 (2015) 1–12. doi:10.1159/000385097. [12] J.Y. Kim, S.C. Park, I. Hwang, H. Cheong, J.W. Nah, K.S. Hahm, Y. Park, Protease inhibitors from plants with antimicrobial activity, Int. J. Mol. Sci. 10 (2009) 2860– 2872. doi:10.3390/ijms10062860. [13] T.N. Shamsi, R. Parveen, S. Fatima, Characterization, biomedical and agricultural applications of protease inhibitors: A review, Int. J. Biol. Macromol. 91 (2016) 1120–1133. doi:10.1016/j.ijbiomac.2016.02.069. [14] C.A. Ryan, Protease Inhibitors in Plants: Genes for Improving Defenses Against Insects and Pathogens, Annu. Rev. Phytopathol. 28 (1990) 425–449. doi:10.1146/annurev.py.28.090190.002233. [15] W. BODE, R. HUBER, Natural protein proteinase inhibitors and their interaction with proteinases, Eur. J. Biochem. 204 (1992) 433–451. doi:10.1111/j.14321033.1992.tb16654.x. [16] M.J. Laskowski, I. Kato, Protein inhibitors of proteinases., Annu. Rev. Biochem. 49 (1980) 593–626. doi:10.1146/annurev.bi.49.070180.003113. [17] B.C. Laskowski, R.L. Jaffe, A. Komornicki, Ab initio calculations of phenylene ring motions in polyphenylene oxide, Int. J. Quantum Chem. 29 (1986) 563–578. doi:10.1002/qua.560290328. [18] K.M. Heutinck, I.J.M. ten Berge, C.E. Hack, J. Hamann, A.T. Rowshani, Serine proteases of the human immune system in health and disease, Mol. Immunol. 47 (2010) 1943–1955. doi:10.1016/j.molimm.2010.04.020. [19] A. Udvardy, The role of controlled proteolysis in cellcycle regulation., Eur. J. Biochem. 240 (1996) 307– 313. doi:10.1111/j.1432-1033.1996.0307h.x. [20] N. Behrendt, Matrix Proteases in Health and Disease, 2012. https://scholar.google.com/scholar_lookup?title=Matr ix+Proteases+in+Health+and+Disease&publication_yea r=2012&. [21] J.A. Torres-Castillo, C.M. Jacobo, A. Blanco-Labra, Characterization of a highly stable trypsin-like 35

Jo

ur na

lP

re

-p

ro

of

proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire), Phytochemistry. 70 (2009) 1374–1381. doi:10.1016/j.phytochem.2009.08.009. [22] M.L.R. MacEdo, E.B.S. Diz Filho, M.G.M. Freire, M.L. V. Oliva, J.T. Sumikawa, M.H. Toyama, S. Marangoni, A trypsin inhibitor from Sapindus saponaria L. seeds: Purification, characterization, and activity towards pest insect digestive enzyme, Protein J. 30 (2011) 9– 19. doi:10.1007/s10930-010-9296-7. [23] C. Souza, R. Camargo, M. Demasi, J.M. Santana, Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells, 9 (2014) 1–10. doi:10.1371/journal.pone.0086600. [24] J.E. Monteiro Júnior, N.F. Valadares, H.D.M. Pereira, F.H. Dyszy, A.J. da Costa Filho, A.F. Uchôa, A.S. de Oliveira, C.P. da Silveira Carvalho, T.B. Grangeiro, Expression in Escherichia coli of cysteine protease inhibitors from cowpea (Vigna unguiculata): The crystal structure of a single-domain cystatin gives insights on its thermal and pH stability, Int. J. Biol. Macromol. 102 (2017) 29–41. doi:10.1016/j.ijbiomac.2017.04.008. [25] J. Cotabarren, M.E. Tellechea, F.X. Avilés, J. Lorenzo Rivera, W.D. Obregón, Biochemical characterization of the YBPCI miniprotein, the first carboxypeptidase inhibitor isolated from Yellow Bell Pepper (Capsicum annuum L). A novel contribution to the knowledge of miniproteins stability, Protein Expr. Purif. 144 (2018) 55–61. doi:10.1016/j.pep.2017.12.003. [26] T.F. Martins, I.M. Vasconcelos, R.G.G. Silva, F.D.A. Silva, P.F.N. Souza, A.L.N. Varela, L.M. Albuquerque, J.T.A. Oliveira, A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage, J. Nat. Prod. 81 (2018) 1497–1507. doi:10.1021/acs.jnatprod.7b00545. [27] O.P. Ward, M.B. Rao, A. Kulkarni, Proteases, Production, Encycl. Microbiol. (2009) 495–511. [28] S. Srikanth, Z. Chen, Plant Protease Inhibitors in Therapeutics-Focus on Cancer Therapy, 7 (2016). 36

Jo

ur na

lP

re

-p

ro

of

doi:10.3389/fphar.2016.00470. [29] A. Kobayashi, M.-I. Kang, Y. Watai, K.I. Tong, T. Shibata, K. Uchida, M. Yamamoto, Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1., Mol. Cell. Biol. 26 (2006) 221–229. doi:10.1128/MCB.26.1.221-229.2006. [30] D.A. Lichtenstein, G.A. Meziere, Relevance of lung ultrasound in the diagnosis of acute respiratory failure: the BLUE protocol., Chest. 134 (2008) 117– 125. doi:10.1378/chest.07-2800. [31] S. Kobayashi, K. Asakura, H. Suga, S. Sasaki, High protein intake is associated with low prevalence of frailty among old Japanese women: a multicenter cross-sectional study., Nutr. J. 12 (2013) 164. doi:10.1186/1475-2891-12-164. [32] S. Barberis, H.G. Quiroga, C. Barcia, J.M. Talia, N. Debattista, Natural Food Preservatives Against Microorganisms, Elsevier Inc., 2018. doi:10.1016/B978-0-12-814956-0.00020-2. [33] M.H. Kim, S.C. Park, J.Y. Kim, S.Y. Lee, H.T. Lim, H. Cheong, K.S. Hahm, Y. Park, Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety “Golden Valley,” Biochem. Biophys. Res. Commun. 346 (2006) 681–686. doi:10.1016/j.bbrc.2006.05.186. [34] M.L.R. Macedo, S.F.F. Ribeiro, G.B. Taveira, V.M. Gomes, K.M.C.A. de Barros, S. Maria-Neto, Antimicrobial Activity of ILTI, a Kunitz‐ Type Trypsin Inhibitor from Inga laurina (SW.) Willd, Curr. Microbiol. 72 (2016) 538–544. doi:10.1007/s00284015-0970-z. [35] S.H. Habib, H. Kausar, H.M. Saud, Plant GrowthPromoting Rhizobacteria Enhance Salinity Stress Tolerance in Okra through ROS-Scavenging Enzymes, Biomed Res. Int. 2016 (2016). doi:10.1155/2016/6284547. [36] A. Singh, S. Benjakul, Proteolysis and Its Control Using Protease Inhibitors in Fish and Fish Products: A Review, Compr. Rev. Food Sci. Food Saf. 17 (2018) 496–509. doi:10.1111/1541-4337.12337. [37] F. Part III, Proteases in fish and shellfish: Role on muscle softening and prevention, Int. Food Res. J. 21 37

Jo

ur na

lP

re

-p

ro

of

(2014) 433–445. [38] B. Bijina, S. Chellappan, S.M. Basheer, K.K. Elyas, A.H. Bahkali, M. Chandrasekaran, Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization, Process Biochem. 46 (2011) 2291– 2300. doi:10.1016/j.procbio.2011.09.008. [39] F.J. Alarcón, F.L. García-Carreño, M.A. Navarrete Del Toro, Effect of plant protease inhibitors on digestive proteases in two fish species, Lutjanus argentiventris and L. novemfasciatus, Fish Physiol. Biochem. 24 (2001) 179–189. doi:10.1023/A:1014079919461. [40] T.N. Shamsi, R. Parveen, S. Afreen, M. Azam, P. Sen, Y. Sharma, Q. Mohd, R. Haque, T. Fatma, Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant , Anti- Inflammatory , and Antibacterial Activity, 0211 (2018). doi:10.1080/19390211.2017.1407383. [41] B. Pignol, S. Auvin, D. Carré, J.G. Marin, P.E. Chabrier, Calpain inhibitors and antioxidants act synergistically to prevent cell necrosis: Effects of the novel dual inhibitors (cysteine protease inhibitor and antioxidant) BN 82204 and its pro-drug BN 82270, J. Neurochem. 98 (2006) 1217–1228. doi:10.1111/j.1471-4159.2006.03952.x. [42] T.A. Valueva, V. V. Mosolov, Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms, Biochem. 69 (2004) 1305–1309. doi:10.1007/s10541-005-0015-5. [43] P. K. Lawrence, K. Ram Koundal, Plant protease inhibitors in control of phytophagous insects, 2002. doi:10.4067/S0717-34582002000100014. [44] S. Mandal, P. Kundu, B. Roy, R.K. Mandal, Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor , J. Biol. Chem. 277 (2002) 37161–37168. doi:10.1074/jbc.m205280200. [45] F. De Leo, M. Volpicella, F. Licciulli, S. Liuni, R. Gallerani, R. Ceci, PLANT-PIs: a database for plant protease inhibitors and their genes, Nucleic Acids Res. 30 (2002) 347–348. doi:10.1093/nar/30.1.347. [46] J. Sels, J. Mathys, B.M.A. De Coninck, B.P.A. Cammue, M.F.C. De Bolle, Plant pathogenesis-related (PR) 38

Jo

ur na

lP

re

-p

ro

of

proteins: a focus on PR peptides., Plant Physiol. Biochem. PPB. 46 (2008) 941–950. doi:10.1016/j.plaphy.2008.06.011. [47] J. Brzin, M. Kidrič, Proteinases and their inhibitors in plants: Role in normal growth and in response to various stress conditions, Biotechnol. Genet. Eng. Rev. 13 (1996) 421–468. doi:10.1080/02648725.1996.10647936. [48] Z.F. Xu, W.Q. Qi, X.Z. Ouyang, E. Yeung, M.L. Chye, A proteinase inhibitor II of Solanum americanum is expressed in phloem., Plant Mol. Biol. 47 (2001) 727– 738. [49] S. Sin, M. Chye, Expression of proteinase inhibitor II proteins during floral development in Solanum americanum, (2004) 1010–1022. doi:10.1007/s00425004-1306-6. [50] G.C. Mello, M.L. V Oliva, J.T. Sumikawa, O.L.T. Machado, S. Marangoni, J.C. Novello, M.L.R. Macedo, Purification and Characterization of a New Trypsin Inhibitor from Dimorphandra mollis Seeds, 20 (2002). [51] S.K. Haq, R.H. Khan, Characterization of a Proteinase Inhibitor from Cajanus cajan ( L .), 22 (2003). [52] S.-G. Fan, G.-J. Wu, Characteristics of plant proteinase inhibitors and their applications in combating phytophagous insects, Bot. Bull. Acad. Sin. 46 (2005) 273–292. doi:10.1007/978-3-540-73589-2_15. [53] S.K. Haq, S.M. Atif, R.H. Khan, Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection, 431 (2004) 145–159. doi:10.1016/j.abb.2004.07.022. [54] A.D. Bendre, S. Ramasamy, C.G. Suresh, Analysis of Kunitz inhibitors from plants for comprehensive structural and functional insights, Int. J. Biol. Macromol. 113 (2018) 933–943. doi:10.1016/j.ijbiomac.2018.02.148. [55] R.-F. Qi, Z.-W. Song, C.-W. Chi, Structural features and molecular evolution of Bowman-Birk protease inhibitors and their potential application., Acta Biochim. Biophys. Sin. (Shanghai). 37 (2005) 283–292. [56] L. Chiche, A. Heitz, J.-C. Gelly, J. Gracy, P.T.T. Chau, P.T. Ha, J.-F. Hernandez, D. Le-Nguyen, Squash inhibitors: from structural motifs to macrocyclic 39

Jo

ur na

lP

re

-p

ro

of

knottins., Curr. Protein Pept. Sci. 5 (2004) 341–349. [57] L. Chiche, G. Postic, P. Charlotte, J. Gelly, KNOTTIN  : the database of inhibitor cystine knot scaffold after 10 years , toward a systematic structure modeling, 46 (2018) 454–458. doi:10.1093/nar/gkx1084. [58] M. Benchabane, U. Schlüter, J. Vorster, M.C. Goulet, D. Michaud, Plant cystatins, Biochimie. 92 (2010) 1657– 1666. doi:10.1016/j.biochi.2010.06.006. [59] M. Martinez, Z. Abraham, M. Gambardella, M. Echaide, P. Carbonero, I. Diaz, The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties., J. Exp. Bot. 56 (2005) 1821–1829. doi:10.1093/jxb/eri172. [60] T.A. Walsh, J.A. Strickland, Proteolysis of the 85kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains., Plant Physiol. 103 (1993) 1227–1234. [61] K. V Fernandes, P.A. Sabelli, D.H. Barratt, M. Richardson, J. Xavier-Filho, P.R. Shewry, The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors., Plant Mol. Biol. 23 (1993) 215–219. [62] C. Waldron, L.M. Wegrich, P.A. Merlo, T.A. Walsh, Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor., Plant Mol. Biol. 23 (1993) 801– 812. [63] C.O. Lim, S.I. Lee, W.S. Chung, S.H. Park, I. Hwang, M.J. Cho, Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds., Plant Mol. Biol. 30 (1996) 373–379. [64] B.L. Rogersa, J. Pollock, D.G. Klapperb, I.J. Griffith, Sequence of the proteinase-inhibitor cystatin homologue from the pollen of Ambrosia artemisilfolia ( short ragweed )*, 133 (1993) 219–221. [65] A. Ojima, H. Shiota, K. Higashi, H. Kamada, Y. Shimma, M. Wada, S. Satoh, An extracellular insoluble inhibitor of cysteine proteinases in cell cultures and seeds of carrot, (1997) 99–109. [66] I. Song, M. Taylor, K. Baker, R.C.J. Bateman, Inhibition of cysteine proteinases by Carica papaya cystatin 40

Jo

ur na

lP

re

-p

ro

of

produced in Escherichia coli., Gene. 162 (1995) 221– 224. [67] S.N. Ryan, W.A. Laing, M.T. McManus, A cysteine proteinase inhibitor purified from apple fruit., Phytochemistry. 49 (1998) 957–963. [68] M. Kimura, T. Ikeda, D. Fukumoto, N. Yamasaki, M. Yonekura, Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill)., Biosci. Biotechnol. Biochem. 59 (1995) 2328– 2329. [69] B.J. Connors, N.P. Laun, C.A. Maynard, W.A. Powell, Molecular characterization of a gene encoding a cystatin expressed in the stems of American chestnut (Castanea dentata)., Planta. 215 (2002) 510–514. doi:10.1007/s00425-002-0782-9. [70] K.-I. Yoza, S. Nakamura, M. Yaguchi, K. Haraguchi, K.-I. Ohtsubo, Molecular cloning and functional expression of cDNA encoding a cysteine proteinase inhibitor, cystatin, from Job’s tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)., Biosci. Biotechnol. Biochem. 66 (2002) 2287–2291. [71] M. Kuroda, T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N.K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Furuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, K. Hiramatsu, Whole genome sequencing of meticillin-resistant Staphylococcus aureus., Lancet (London, England). 357 (2001) 1225–1240. doi:10.1016/s0140-6736(00)04403-2. [72] S.K. Haq, S.M. Atif, R.H. Khan, Biochemical characterization, stability studies and N-terminal sequence of a bi-functional inhibitor from Phaseolus aureus Roxb. (Mung bean), Biochimie. 87 (2005) 1127–1136. doi:10.1016/j.biochi.2005.05.007. [73] M. Mares, B. Meloun, M. Pavlik, V. Kostka, M. Baudys, Primary structure of cathepsin D inhibitor from potatoes and its structure relationship to soybean trypsin inhibitor family., FEBS Lett. 251 (1989) 94–98. 41

Jo

ur na

lP

re

-p

ro

of

[74] J. Kervinen, G.J. Tobin, J. Costa, D.S. Waugh, A. Wlodawer, A. Zdanov, Crystal structure of plant aspartic proteinase prophytepsin: inactivation and vacuolar targeting., EMBO J. 18 (1999) 3947–3955. doi:10.1093/emboj/18.14.3947. [75] H. Park, N. Yamanaka, A. Mikkonen, I. Kusakabe, H. Kobayashi, Purification and characterization of aspartic proteinase from sunflower seeds., Biosci. Biotechnol. Biochem. 64 (2000) 931–939. [76] S.A. Cater, W.E. Lees, J. Hill, J. Brzin, J. Kay, L.H. Phylip, Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D., Biochim. Biophys. Acta. 1596 (2002) 76– 82. [77] D.C. Rees, W.N. Lipscomb, Refined crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 A resolution., J. Mol. Biol. 160 (1982) 475–498. [78] J.L. Arolas, J. Vendrell, F.X. Aviles, L.D. Fricker, Metallocarboxypeptidases: emerging drug targets in biomedicine., Curr. Pharm. Des. 13 (2007) 349–366. [79] D. Lufrano, J. Cotabarren, J. Garcia-Pardo, R. Fernandez-Alvarez, O. Tort, S. Tanco, F.X. Avil??s, J. Lorenzo, W.D. Obreg??n, Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes, Phytochemistry. 120 (2015) 36– 45. doi:10.1016/j.phytochem.2015.09.010. [80] J. Cotabarren, M.E. Tellechea, S.M. Tanco, J. Lorenzo, J. Garcia-Pardo, F.X. Avilés, W.D. Obregón, Biochemical and MALDI-TOF mass spectrometric characterization of a novel native and recombinant cystine knot miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña, Int. J. Mol. Sci. 19 (2018) 1–19. doi:10.3390/ijms19030678. [81] G.M. Hass, H. Nau, K. Biemann, D.T. Grahn, L.H. Ericsson, H. Neurath, The amino acid sequence of a carboxypeptidase inhibitor from potatoes., Biochemistry. 14 (1975) 1334–1342. [82] J.S. Graham, C.A. Ryan, Accumulation of a metallocarboxypeptidase inhibitor in leaves of wounded potato plants., Biochem. Biophys. Res. Commun. 101 (1981) 1164–1170. [83] M. a. Jongsma, C. Bolter, The adaptation of insects to 42

Jo

ur na

lP

re

-p

ro

of

plant protease inhibitors., J. Insect Physiol. 43 (1997) 885–895. doi:10.1016/s0022-1910(97)00040-1. [84] V.A. Hilder, A.M.R. Gatehoued, D. Boulter, Transgenic plants conferring insect tolerance: Protease inhibitor approach, Eng. Util. (1993) 317–337. [85] R. Gutierrez-campos, J.A. Torres-acosta, L.J. Saucedoarias, M.A. Gomez-lim, The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants, (2000). doi:10.1038/70781. [86] K.M. Dunse, J.A. Stevens, F.T. Lay, Y.M. Gaspar, R.L. Heath, M.A. Anderson, Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field., Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 15011–15015. doi:10.1073/pnas.1009241107. [87] K. Zhu-Salzman, R. Zeng, Insect Response to Plant Defensive Protease Inhibitors, Annu. Rev. Entomol. 60 (2015) 233–252. doi:10.1530/REP-16-0083. [88] V. Bobbarala, P.K. Katikala, K.C. Naidu, S. Penumajji, Antifungal activity of selected plants extracts against phytopathogenic fungi Aspergillus niger, Indian J. Sci. Technol. (2009) 87–90. [89] T.N. Shamsi, R. Parveen, A. Ahmad, R.R. Samal, S. Kumar, S. Fatima, Inhibition of gut proteases and development of dengue vector, Aedes aegypti by Allium sativum protease inhibitor, Acta Ecol. Sin. 38 (2018) 325–328. doi:10.1016/j.chnaes.2018.01.002. [90] A. Cingel, J. Savić, J. Lazarević, T. Ćosić, M. Raspor, A. Smigocki, S. Ninković, Co-expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development, Insect Sci. 24 (2017) 768–780. doi:10.1111/1744-7917.12364. [91] J. Quilis, B. López-García, D. Meynard, E. Guiderdoni, B. San Segundo, Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice, Plant Biotechnol. J. 12 (2014) 367–377. doi:10.1111/pbi.12143. [92] J. Quilis, D. Meynard, L. Vila, F.X. Avilés, E. Guiderdoni, B. San Segundo, A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice, 43

Jo

ur na

lP

re

-p

ro

of

Plant Biotechnol. J. 5 (2007) 537–553. doi:10.1111/j.1467-7652.2007.00264.x. [93] A. Bayés, M.R. de la Vega, J. Vendrell, F.X. Aviles, M.A. Jongsma, J. Beekwilder, Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B, Insect Biochem. Mol. Biol. 36 (2006) 654–664. doi:10.1016/j.ibmb.2006.05.010. [94] G.F. Rocha, M.E. Díaz, A.M. Rosso, M.G. Parisi, Citotoxicidad de una aspartil peptidasa de Salpichroa origanifolia frente a la infección causada por Phytophthora capsici en zapallitos verdes (Cucurbita maxima, var. Zapallito), Cultiv. Trop. 37 (2016) 111– 117. doi:10.13140/RG.2.1.4562.3926. [95] I. Smid, A. Rotter, K. Gruden, J. Brzin, M.B. Gasparic, J. Kos, J. Zel, J. Sabotic, Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases, Pestic. Biochem. Physiol. 122 (2015) 59–66. doi:10.1016/j.jalz.2013.05.1647. [96] J. Sabotič, J. Kos, Microbial and fungal protease inhibitors - Current and potential applications, Appl. Microbiol. Biotechnol. 93 (2012) 1351–1375. doi:10.1007/s00253-011-3834-x. [97] J.-C. Gelly, The KNOTTIN website and database: a new information system dedicated to the knottin scaffold, Nucleic Acids Res. 32 (2003) 156D–159. doi:10.1093/nar/gkh015. [98] J.F. Lindahl, D. Grace, The consequences of human actions on risks for infectious diseases: a review, Infect. Ecol. Epidemiol. 5 (2015) 30048. doi:10.3402/iee.v5.30048. [99] Orientaciones de la OMS para proteger la salud frente al cambio climático mediante la planifi cación de la adaptación de la salud, n.d. [100] D.J. Newman, G.M. Cragg, Natural Products as Sources of New Drugs over the Last 25 Years, J. Nat. Prod. 70 (2007) 461–477. doi:10.1021/np068054v. [101] D.J. Newman, G.M. Cragg, Natural Products As Sources of New Drugs over the 30 Years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311–335. 44

Jo

ur na

lP

re

-p

ro

of

doi:10.1021/np200906s. [102] J.E. Ahn, R.A. Salzman, S.C. Braunagel, H. Koiwa, K. Zhu-Salzman, Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor, Insect Mol. Biol. 13 (2004) 649–657. doi:10.1111/j.0962-1075.2004.00523.x. [103] C. Imada, Enzyme inhibitors and other bioactive compounds from marine actinomycetes, Antonie van Leeuwenhoek, Int. J. Gen. Mol. Microbiol. 87 (2005) 59–63. doi:10.1007/s10482-004-6544-x. [104] R.A. Copeland, Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists., Methods Biochem. Anal. 46 (2005) 1–265. [105] R. Cyran, New developments in therapeutic enzyme inhibitors and blockers, Bus. Commun. Co. (2002). [106] J.I. Ottaviani, L. Actis-goretta, J.J. Villordo, C.G. Fraga, Procyanidin structure defines the extent and specificity of angiotensin I converting enzyme inhibition, 88 (2006) 359–365. doi:10.1016/j.biochi.2005.10.001. [107] E. Wood, R. S Hogg, B. Yip, M. V O’Shaughnessy, J. S.G. Montaner, CD4 Cell Count Response to Nonnucleoside Reverse Transcriptase Inhibitor- or Protease Inhibitor-Based Highly Active Antiretroviral Therapy in an Observational Cohort Study, 2003. doi:10.1097/00126334-200311010-00016. [108] J.R. Arribas, F. Pulido, R. Delgado, A. Lorenzo, P. Miralles, A. Arranz, J.J. Gonzalez-Garcia, C. Cepeda, R. Hervas, J.R. Pano, F. Gaya, A. Carcas, M.L. Montes, J.R. Costa, J.M. Pena, Lopinavir/ritonavir as single-drug therapy for maintenance of HIV-1 viral suppression: 48-week results of a randomized, controlled, openlabel, proof-of-concept pilot clinical trial (OK Study)., J. Acquir. Immune Defic. Syndr. 40 (2005) 280–287. [109] S.L. Binford, F. Maldonado, M.A. Brothers, P.T. Weady, L.S. Zalman, J.W. 3rd Meador, D.A. Matthews, A.K. Patick, Conservation of amino acids in human rhinovirus 3C protease correlates with broadspectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor., Antimicrob. 45

Jo

ur na

lP

re

-p

ro

of

Agents Chemother. 49 (2005) 619–626. doi:10.1128/AAC.49.2.619-626.2005. [110] D. Lamarre, P.C. Anderson, M. Bailey, P. Beaulieu, G. Bolger, P. Bonneau, M. Bos, D.R. Cameron, M. Cartier, M.G. Cordingley, A.-M. Faucher, N. Goudreau, S.H. Kawai, G. Kukolj, L. Lagace, S.R. LaPlante, H. Narjes, M.-A. Poupart, J. Rancourt, R.E. Sentjens, R. St George, B. Simoneau, G. Steinmann, D. Thibeault, Y.S. Tsantrizos, S.M. Weldon, C.-L. Yong, M. Llinas-Brunet, An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus., Nature. 426 (2003) 186–189. doi:10.1038/nature02099. [111] B.A. Malcolm, R. Liu, F. Lahser, S. Agrawal, B. Belanger, N. Butkiewicz, R. Chase, F. Gheyas, A. Hart, D. Hesk, P. Ingravallo, C. Jiang, R. Kong, J. Lu, J. Pichardo, A. Prongay, A. Skelton, X. Tong, S. Venkatraman, E. Xia, V. Girijavallabhan, F.G. Njoroge, SCH 503034, a mechanism-based inhibitor of hepatitis C virus NS3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells., Antimicrob. Agents Chemother. 50 (2006) 1013–1020. doi:10.1128/AAC.50.3.1013-1020.2006. [112] A. Clemente, M. Del Carmen Arques, BowmanBirk inhibitors from legumes as colorectal chemopreventive agents, World J. Gastroenterol. 20 (2014) 10305–10315. doi:10.3748/wjg.v20.i30.10305. [113] V. Turk, J. Kos, B. Turk, Turk V, KosJ and Turk B. (2004). Cancer Cell, 5, 409–410, Cancer Cell. 5 (2004) 409–410. [114] A.R. Kennedy, X.S. Wan, Effects of the BowmanBirk inhibitor on growth, invasion, and clonogenic survival of human prostate epithelial cells and prostate cancer cells*, Prostate. 50 (2002) 125–133. doi:10.1002/pros.10041. [115] C. Dash, A. Kulkarni, B. Dunn, M. Rao, Aspartic peptidase inhibitors: implications in drug development., Crit. Rev. Biochem. Mol. Biol. 38 (2003) 89–119. doi:10.1080/713609213. [116] J.G.H. Ruseler-van Embden, L.M.C. van Lieshout, S.A. Smits, I. van Kessel, J.D. Laman, Potato tuber proteins efficiently inhibit human faecal proteolytic 46

Jo

ur na

lP

re

-p

ro

of

activity: implications for treatment of peri-anal dermatitis., Eur. J. Clin. Invest. 34 (2004) 303–311. doi:10.1111/j.1365-2362.2004.01330.x. [117] S.L. Johnson, M. Pellecchia, Structure- and fragment-based approaches to protease inhibition., Curr. Top. Med. Chem. 6 (2006) 317–329. [118] R.J.A. Machado, N.K. V Monteiro, L. Migliolo, O.N. Silva, L. Franco, S. Kiyota, M.P. Bemquerer, M.F.S. Pinto, A.S. Oliveira, A.F. Uchoa, A.H.A. Morais, E.A. Santos, Characterization and Pharmacological Properties of a Novel Multifunctional Kunitz Inhibitor from Erythrina velutina Seeds, 8 (2013). doi:10.1371/journal.pone.0063571. [119] G.J. Huang, Y.L. Ho, H.J. Chen, Y.S. Chang, S.S. Huang, H.J. Hung, Y.H. Lin, Sweet potato storage root trypsin inhibitor and their peptic hydrolysates exhibited angiotensin converting enzyme inhibitory activity in vitro, Bot. Stud. 49 (2008) 101–108. [120] A. Shamsi, B. Bano, Journey of cystatins from being mere thiol protease inhibitors to at heart of many pathological conditions, Int. J. Biol. Macromol. 102 (2017) 674–693. doi:10.1016/j.ijbiomac.2017.04.071. [121] M. Bakail, F. Ochsenbein, Targeting proteinprotein interactions, a wide open field for drug design, Comptes Rendus Chim. 19 (2016) 19–27. doi:10.1016/j.crci.2015.12.004. [122] R.G.G. Silva, I.M. Vasconcelos, A.J.U.B. Filho, A.F.U. Carvalho, T.M. Souza, D.M.F. Gondim, A.L.N. Varela, J.T.A. Oliveira, Castor bean cake contains a trypsin inhibitor that displays antifungal activity against Colletotrichum gloeosporioides and inhibits the midgut proteases of the dengue mosquito larvae, Ind. Crops Prod. 70 (2015) 48–55. doi:10.1016/j.indcrop.2015.02.058. [123] H.P.S. Costa, J.T.A. Oliveira, D.O.B. Sousa, J.K.S. Morais, F.B. Moreno, A.C.O. Monteiro-Moreira, R.A. Viegas, I.M. Vasconcelos, JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment, Front. Microbiol. 5 (2014) 1–12. doi:10.3389/fmicb.2014.00005. 47

Jo

ur na

lP

re

-p

ro

of

[124] H. Habib, K.M. Fazili, Plant protease inhibitors  : a defense strategy in plants, 2 (2007) 68–85. [125] M.S. De Brito, M.B. Melo, J. Perdigão, D.A. Alves, Partial purification of trypsin / papain inhibitors from Hymenaea courbaril L . seeds and antibacterial effect of protein fractions, 43 (2016) 11–18. [126] Y.-R. Zhao, Y.-H. Xu, H.-S. Jiang, S. Xu, X.-F. Zhao, J.-X. Wang, Antibacterial activity of serine protease inhibitor 1 from kuruma shrimp Marsupenaeus japonicus., Dev. Comp. Immunol. 44 (2014) 261–269. doi:10.1016/j.dci.2014.01.002. [127] L.S. Satheesh, K. Murugan, Antimicrobial activity of protease inhibitor from leaves of Coccinia grandis ( L .) Voigt ., 49 (2011) 366–374. [128] S. R, P. R, Antimicrobial Activity of a Protease Inhibitor Isolated From the Rhizome of Curcuma Amada, Asian J. Pharm. Clin. Res. 10 (2017) 131. doi:10.22159/ajpcr.2017.v10i9.18257. [129] E. Montesinos, Antimicrobial peptides and plant disease control, FEMS Microbiol. Lett. 270 (2007) 1– 11. doi:10.1111/j.1574-6968.2007.00683.x. [130] Y. Park, B.H. Choi, J.-S. Kwak, C.-W. Kang, H.-T. Lim, H.-S. Cheong, K.-S. Hahm, Kunitz-type serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung)., J. Agric. Food Chem. 53 (2005) 6491– 6496. doi:10.1021/jf0505123. [131] J. Pandhare, K. Zog, V. V. Deshpande, Differential stabilities of alkaline protease inhibitors from actinomycetes: Effect of various additives on thermostability, Bioresour. Technol. 84 (2002) 165– 169. doi:10.1016/S0960-8524(02)00025-1. [132] J.L. Martinez Vidal, P. Plaza-Bolanos, R. RomeroGonzalez, A. Garrido Frenich, Determination of pesticide transformation products: a review of extraction and detection methods., J. Chromatogr. A. 1216 (2009) 6767–6788. doi:10.1016/j.chroma.2009.08.013. [133] A.C. Belfroid, M. van Drunen, M.A. Beek, S.M. Schrap, C.A. van Gestel, B. van Hattum, Relative risks of transformation products of pesticides for aquatic ecosystems., Sci. Total Environ. 222 (1998) 167–183. [134] I. Cavoski, P. Caboni, G. Sarais, T. Miano, 48

Jo

ur na

lP

re

-p

ro

of

Degradation and persistence of rotenone in soils and influence of temperature variations., J. Agric. Food Chem. 56 (2008) 8066–8073. doi:10.1021/jf801461h. [135] J. Lu, L. Wu, J. Newman, B. Faber, J. Gan, Degradation of pesticides in nursery recycling pond waters., J. Agric. Food Chem. 54 (2006) 2658–2663. doi:10.1021/jf060067r. [136] A.M.A. van der Linden, A. Tiktak, J.J.T.I. Boesten, A. Leijnse, Influence of pH-dependent sorption and transformation on simulated pesticide leaching., Sci. Total Environ. 407 (2009) 3415–3420. doi:10.1016/j.scitotenv.2009.01.059. [137] V. Andreu, Y. Pico, Determination of Pesticides and their Degradation Products in Soil: Critical Review and Comparison of Methods, 2004. doi:10.1016/j.trac.2004.07.008. [138] F. Jamal, P.K. Pandey, D. Singh, M.Y. Khan, Serine protease inhibitors in plants: Nature’s arsenal crafted for insect predators, Phytochem. Rev. 12 (2013) 1–34. doi:10.1002/asjc.1321. [139] F. Jamal, P.K. Pandey, D. Singh, W. Ahmed, A Kunitz-type serine protease inhibitor from Butea monosperma seed and its influence on developmental physiology of Helicoverpa armigera, Process Biochem. 50 (2015) 311–316. doi:10.1016/j.procbio.2014.12.003. [140] E. Nathala, S. Dhingra, Biological Effects of Caesalpinia crista Seed Extracts on Helicoverpa armigera (Lepidoptera: Noctuidae) and Its Predator, Coccinella septumpunctata (Coleoptera: Coccinellidae), J. Asia. Pac. Entomol. 9 (2006) 159– 164. doi:10.1016/S1226-8615(08)60287-3. [141] D. Yang, L. Chai, J. Wang, X. Zhao, Molecular cloning and characterization of Hearm caspase-1 from Helicoverpa armigera, Mol. Biol. Rep. 35 (2008) 405– 412. doi:10.1007/s11033-007-9100-8. [142] J.A. Stevens, K.M. Dunse, R.F. Guarino, B.L. Barbeta, S.C. Evans, J.A. West, M.A. Anderson, The impact of ingested potato type II inhibitors on the production of the major serine proteases in the gut of Helicoverpa armigera, Insect Biochem. Mol. Biol. 43 (2013) 197–208. doi:10.1016/j.ibmb.2012.11.006. 49

Jo

ur na

lP

re

-p

ro

of

[143] Y. Ge, Y.M. Cai, L. Bonneau, V. Rotari, A. Danon, E.A. McKenzie, H. McLellan, L. MacH, P. Gallois, Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis, Cell Death Differ. 23 (2016) 1493–1501. doi:10.1038/cdd.2016.34. [144] K. Sharma, Protease inhibitors in crop protection from insects, Int J Curr Res Aca Rev. 3 (2015) 55–70. [145] M. V. Padul, R.D. Tak, M.S. Kachole, Protease inhibitor (PI) mediated defense in leaves and flowers of pigeonpea (protease inhibitor mediated defense in pigeonpea), Plant Physiol. Biochem. 52 (2012) 77–82. doi:10.1016/j.plaphy.2011.10.018. [146] V.G. Meenu Krishnan, K. Murugan, Purification, characterization and kinetics of protease inhibitor from fruits of Solanum aculeatissimum Jacq, Food Sci. Hum. Wellness. 4 (2015) 97–107. doi:10.1016/j.fshw.2015.06.003. [147] F.M. Grosse-Holz, R.A.L. van der Hoorn, Juggling jobs: Roles and mechanisms of multifunctional protease inhibitors in plants, New Phytol. 210 (2016) 794–807. doi:10.1111/nph.13839. [148] B.N. Joshi, M.N. Sainani, K.B. Bastawade, V.S. Gupta, P.K. Ranjekar, Cysteine protease inhibitor from pearl millet: A new class of antifungal protein, Biochem. Biophys. Res. Commun. 246 (1998) 382–387. doi:10.1006/bbrc.1998.8625. [149] R.H. Shukle, L. Wu, The Role of Protease Inhibitors and Parasitoids on the Population Dynamics of Sitotroga cerealella (Lepidoptera: Gelechiidae), Environ. Entomol. 32 (2003) 488–499. https://doi.org/10.1603/0046-225X-32.3.488. [150] D.P. Bown, H.S. Wilkinson, J.A. Gatehouse, Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families, Insect Biochem. Mol. Biol. 27 (1997) 625–638. doi:10.1016/S09651748(97)00043-X. [151] A.M. Harsulkar, A.P. Giri, A.G. Patankar, V.S. Gupta, M.N. Sainani, P.K. Ranjekar, V. V. Deshpande, Successive Use of Non-Host Plant Proteinase Inhibitors 50

Jo

ur na

lP

re

-p

ro

of

Required for Effective Inhibition of Helicoverpa armigera Gut Proteinases and Larval Growth 1, Society. 121 (1999) 497–506. doi:10.1104/pp.121.2.497. [152] M.A. Jongsma, W.J. Stiekema, D. Bosch, Combatting inhibitor-insensitive proteases of insect pests, Trends Biotechnol. 14 (1996) 331–333. doi:10.1016/0167-7799(96)30019-X. [153] X. Duan, X. Li, Q. Xue, M. Abo-el-Saad, D. Xu, R. Wu, Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant., Nat. Biotechnol. 14 (1996) 494–498. doi:10.1038/nbt0496-494. [154] V.A. Hilder, A.M.R. Gatehouse, S.E. Sheerman, R.F. Barker, D. Boulter, A novel mechanism of insect resistance engineered into tobacco, Nature. 330 (1987) 160. https://doi.org/10.1038/330160a0. [155] R. Johnson, J. Narvaez, G. An, C. Ryan, Expression of proteinase inhibitors I and II in transgenic tobacco plants: effects on natural defense against Manduca sexta larvae., Proc. Natl. Acad. Sci. 86 (1989) 9871– 9875. doi:10.1073/pnas.86.24.9871. [156] M.L.R. Macedo, C.F.R. de Oliveira, P.M. Costa, E.C. Castelhano, M.C. Silva-Filho, Adaptive mechanisms of insect pests against plant protease inhibitors and future prospects related to crop protection: a review., Protein Pept. Lett. 22 (2015) 149–163. [157] M. Swathi, V. Lokya, V. Swaroop, N. Mallikarjuna, M. Kannan, A. Dutta-Gupta, K. Padmasree, Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118), Plant Physiol. Biochem. 83 (2014) 77–87. doi:10.1016/j.plaphy.2014.07.009. [158] A.P. Kaur, S.K. Sohal, Pea protease inhibitor inhibits protease activity and development of Bactrocera cucurbitae, J. Asia. Pac. Entomol. 19 (2016) 1183–1189. doi:10.1016/j.aspen.2016.10.012. [159] M.L.V. Oliva, R. da S. Ferreira, J.G. Ferreira, C.A.A. de Paula, C.E. Salas, M.U. Sampaio, Structural and functional properties of kunitz proteinase inhibitors from leguminosae: a mini review., Curr. Protein Pept. Sci. 12 (2011) 348–357. 51

Jo

ur na

lP

re

-p

ro

of

[160] S.B. Goodwin, Population Genetics of Soilborne Fungal Plant Pathogens, Phytopathology. 87 (1997) 462–473. [161] J.R. Mendieta, M.R. Pagano, F.F. Muñoz, G.R. Daleo, M.G. Guevara, Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization, Microbiology. 152 (2006) 2039– 2047. doi:10.1099/mic.0.28816-0. [162] M.R. Pagano, J.R. Mendieta, F.F. Muñoz, G.R. Daleo, M.G. Guevara, Roles of glycosylation on the antifungal activity and apoplast accumulation of StAPs (Solanum tuberosum aspartic proteases), Int. J. Biol. Macromol. 41 (2007) 512–520. doi:10.1016/j.ijbiomac.2007.07.009. [163] G.W. Chen, J.G. Chung, H.C. Ho, J.G. Lin, Effects of the garlic compounds diallyl sulphide and diallyl disulphide on arylamine N-acetyltransferase activity in Klebsiella pneumoniae., J. Appl. Toxicol. 19 (1999) 75–81. [164] M.M. Tajkarimi, S.A. Ibrahim, D.O. Cliver, Antimicrobial herb and spice compounds in food, Food Control. 21 (2010) 1199–1218. doi:10.1016/j.foodcont.2010.02.003. [165] S. Ananou, M. Maqueda, M. Martinez-Bueno, A. Galvez, E. Valdivia, Bactericidal synergism through enterocin AS-48 and chemical preservatives against Staphylococcus aureus., Lett. Appl. Microbiol. 45 (2007) 19–23. doi:10.1111/j.1472-765X.2007.02155.x. [166] T.C. Baird-Parker, The production of microbiologically safe and stable foods, 2000. [167] P.M. Davidson, F.J. Critzer, T.M. Taylor, Naturally Occurring Antimicrobials for Minimally Processed Foods, Annu. Rev. Food Sci. Technol. 4 (2013) 163– 190. doi:10.1146/annurev-food-030212-182535. [168] A. Lucera, C. Costa, A. Conte, M.A. Del Nobile, Food applications of natural antimicrobial compounds, Front. Microbiol. 3 (2012) 1–13. doi:10.3389/fmicb.2012.00287. [169] F. Fratianni, F. Nazzaro, A. Marandino, M.D.R. Fusco, R. Coppola, V. De Feo, L. De Martino, Biochemical composition, antimicrobial activities,and anti-quorum-sensing activities of ethanol and ethyl 52

Jo

ur na

lP

re

-p

ro

of

acetate extracts from Hypericum connatum Lam. (Guttiferae)., J. Med. Food. 16 (2013) 454–459. doi:10.1089/jmf.2012.0197. [170] K.D. REPPOND, J.K. BABBITT, Protease Inhibitors Affect Physical Properties of Arrowtooth Flounder and Walleye Pollock Surimi, J. Food Sci. 58 (1993) 96–98. doi:10.1111/j.1365-2621.1993.tb03218.x. [171] C. Zhong, Q.F. Cai, G.M. Liu, L.C. Sun, K. Hara, W.J. Su, M.J. Cao, Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins, Food Chem. 133 (2012) 1560–1568. doi:10.1016/j.foodchem.2012.02.050. [172] Y. ZHANG, Y. KOUZUMA, T. MIYAJI, M. YONEKURA, Purification, Characterization, and cDNA Cloning of a Bowman-Birk Type Trypsin Inhibitor from Apios americana Medikus Tubers, Biosci. Biotechnol. Biochem. 72 (2008) 171–178. doi:10.1271/bbb.70531. [173] R. Roychaudhuri, G. Sarath, M. Zeece, J. Markwell, Reversible denaturation of the soybean Kunitz trypsin inhibitor., Arch. Biochem. Biophys. 412 (2003) 20–26. doi:10.1016/s0003-9861(03)00011-0. [174] H. Liao, W. Ren, Z. Kang, J.H. Jiang, X.J. Zhou, L.F. Du, A trypsin inhibitor from Cassia obtusifolia seeds: isolation, characterization and activity on Pieris rapae., Biotechnol. Lett. 4 (2007) 653–658. [175] L. Yoshizaki, M.F. Troncoso, J.L.S. Lopes, U. Hellman, L.M. Beltramini, C. Wolfenstein-Todel, Calliandra selloi Macbride trypsin inhibitor: Isolation, characterization, stability, spectroscopic analyses, Phytochemistry. 68 (2007) 2625–2634. doi:10.1016/j.phytochem.2007.06.003. [176] J.-Y. Zhou, H. Liao, N.-H. Zhang, L. Tang, Y. Xu, F. Chen, Identification of a Kunitz inhibitor from Albizzia kalkora and its inhibitory effect against pest midgut proteases, Biotechnol. Lett. 30 (2008) 1495—1499. doi:10.1007/s10529-008-9699-0. [177] N.S. Chaudhary, C. Shee, A. Islam, F. Ahmad, D. Yernool, P. Kumar, A.K. Sharma, Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds, Phytochemistry. 69 (2008) 2120– 53

Jo

ur na

lP

re

-p

ro

of

2126. doi:10.1016/j.phytochem.2008.05.002. [178] M. Kidric, H. Fabian, J. Brzin, T. Popovic, R.H. Pain, Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor., Biochem. Biophys. Res. Commun. 297 (2002) 962–967. doi:10.1016/s0006-291x(02)02328-8. [179] V.A. Garcia, M.D.G.M. Freire, J.C. Novello, S. Marangoni, M.L.R. Macedo, Trypsin inhibitor from Poecilanthe parviflora seeds: Purification, characterization, and activity against pest proteases, Protein J. 23 (2004) 343–350. doi:10.1023/B:JOPC.0000032654.67733.d5. [180] C.E.M. Gomes, A.E.A.D. Barbosa, L.L.P. Macedo, J.C.M. Pitanga, F.T. Moura, A.S. Oliveira, R.M. Moura, A.F.S. Queiroz, F.P. Macedo, L.B.S. Andrade, M.S. Vidal, M.P. Sales, Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly), Plant Physiol. Biochem. 43 (2005) 1095–1102. doi:10.1016/j.plaphy.2005.11.004. [181] T.N. Shamsi, R. Parveen, M. Amir, M.A. Baig, M. Irfan Qureshi, S. Ali, S. Fatima, Allium sativum protease inhibitor: A novel kunitz trypsin inhibitor from garlic is a new comrade of the serpin family, PLoS One. 11 (2016) 1–16. doi:10.1371/journal.pone.0165572. [182] S. Prathibha, B. Nambisan, S. Leelamma, Enzyme inhibitors in tuber crops and their thermal stability., Plant Foods Hum. Nutr. 48 (1995) 247–257. [183] V. Lanzotti, The analysis of onion and garlic, J. Chromatogr. A. 1112 (2006) 3–22. doi:10.1016/j.chroma.2005.12.016. [184] C.F.R. De Oliveira, I.M. Vasconcelos, R. Aparicio, M.D.G.M.H. Freire, P.A. Baldasso, S. Marangoni, M.L.R. MacEdo, Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds, Process Biochem. 47 (2012) 929–935. doi:10.1016/j.procbio.2012.02.022. [185] A. Bhattacharyya, S. Rai, C.R. Babu, A trypsin and chymotrypsin inhibitor from Caesalpinia bonduc seeds: Isolation, partial characterization and insecticidal properties, Plant Physiol. Biochem. 45 54

Jo

ur na

lP

re

-p

ro

of

(2007) 169–177. doi:10.1016/j.plaphy.2007.02.003. [186] A. Bhattacharyya, S. Mazumdar, S.M. Leighton, C.R. Babu, A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: Purification, characterization, and kinetic properties, Phytochemistry. 67 (2006) 232–241. doi:10.1016/j.phytochem.2005.11.010. [187] M.F. Siddiqui, A. Ahmed, B. Bano, Insight into the biochemical, kinetic and spectroscopic characterization of garlic (Allium sativum) phytocystatin: Implication for cardiovascular disease, Int. J. Biol. Macromol. 95 (2017) 734–742. doi:10.1016/j.ijbiomac.2016.11.107. [188] A.O.A. El-Latif, Protease purification and characterization of a serine protease inhibitor from Egyptian varieties of soybean seeds and its efficacy against Spodoptera littoralis, J. Plant Prot. Res. 55 (2015) 16–25. doi:10.1515/jppr-2015-0003. [189] A.R. Dabhade, N.U. Mokashe, U.K. Patil, Purification, characterization, and antimicrobial activity of nontoxic trypsin inhibitor from Albizia amara Boiv., Process Biochem. 51 (2016) 659–674. doi:10.1016/j.procbio.2016.02.015. [190] A.O. Abd El-latif, In vivo and in vitro inhibition of Spodoptera littoralis gut-serine protease by protease inhibitors isolated from maize and sorghum seeds, Pestic. Biochem. Physiol. 116 (2014) 40–48. doi:10.1016/j.pestbp.2014.09.009. [191] L.C. Guimarães, C.F.R. de Oliveira, S. Marangoni, D.G.L. de Oliveira, M.L.R. Macedo, Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth, Pestic. Biochem. Physiol. 118 (2015) 1–9. doi:10.1016/j.pestbp.2014.12.001. [192] M. Azarkan, R. Dibiani, E. Goormaghtigh, V. Raussens, D. Baeyens-Volant, The papaya Kunitz-type trypsin inhibitor is a highly stable β-sheet glycoprotein, Biochim. Biophys. Acta - Proteins Proteomics. 1764 (2006) 1063–1072. doi:10.1016/j.bbapap.2006.02.014. [193] W.D. Obregón, N. Ghiano, M. Tellechea, J.S. 55

Jo

ur na

lP

re

-p

ro

of

Cisneros, C.M. Lazza, L.M.I. López, F.X. Avilés, Detection and characterisation of a new metallocarboxypeptidase inhibitor from Solanum tuberosum cv. Desirèe using proteomic techniques, Food Chem. 133 (2012) 1163–1168. doi:10.1016/j.foodchem.2011.08.030. [194] R. Kansal, M. Kumar, K. Kuhar, R.N. Gupta, B. Subrahmanyam, K.R. Koundal, V.K. Gupta, Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera, Brazilian J. Plant Physiol. 20 (2009) 313– 322. doi:10.1590/s1677-04202008000400007. [195] E.F. Fang, J.H. Wong, C.S.F. Bah, P. Lin, S.W. Tsao, T.B. Ng, Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications, Biochem. Biophys. Res. Commun. 396 (2010) 806–811. doi:10.1016/j.bbrc.2010.04.140. [196] A. Ben Bacha, I. Jemel, N.M.S. Moubayed, I. Ben Abdelmalek, Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug, 3 Biotech. 7 (2017) 148. doi:10.1007/s13205-017-0764z. [197] R.M.P. Brandão-Costa, V.F. Araújo, A.L.F. Porto, CgTI, a novel thermostable Kunitz trypsin-inhibitor purified from Cassia grandis seeds: Purification, characterization and termiticidal activity, Int. J. Biol. Macromol. 118 (2018) 2296–2306. doi:10.1016/j.ijbiomac.2018.07.110. [198] H.X. Dib, D.G.L. de Oliveira, C.F.R. de Oliveira, G.B. Taveira, E. de Oliveira Mello, N.V. Verbisk, M.R. Chang, D. Corrêa Junior, V.M. Gomes, M.L.R. Macedo, Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp., Arch. Microbiol. 0 (2018) 0. doi:10.1007/s00203-018-1598-8. [199] P.K. Pandey, D. Singh, R. Singh, M.K. Sinha, S. Singh, F. Jamal, Cassia fistula seed’s trypsin inhibitor(s) as antibiosis agent in Helicoverpa armigera pest management, Biocatal. Agric. Biotechnol. 6 (2016) 202–208. doi:10.1016/j.bcab.2016.04.005. 56

Jo

ur na

lP

re

-p

ro

of

[200] S.R. Ramalho, C.D.S. Bezerra, D.G. Lourenço De Oliveira, L. Souza Lima, S. Maria Neto, C.F. Ramalho De Oliveira, N. Valério Verbisck, M.L. Rodrigues Macedo, Novel Peptidase Kunitz Inhibitor from Platypodium elegans Seeds Is Active against Spodoptera frugiperda Larvae, J. Agric. Food Chem. 66 (2018) 1349–1358. doi:10.1021/acs.jafc.7b04159. [201] M.L.R. Macedo, V.A. Garcia, M. das G.M. Freire, M. Richardson, Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd., Phytochemistry. 68 (2007) 1104–1111. doi:10.1016/j.phytochem.2007.01.024. [202] V.R. Tripathi, A.A. Sahasrabuddhe, S. Kumar, S.K. Garg, Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer, Process Biochem. 49 (2014) 347–355. doi:10.1016/j.procbio.2013.11.007. [203] L.P. Dias, J.T.A. Oliveira, L.C.B. Rocha-Bezerra, D.O.B. Sousa, H.P.S. Costa, N.M.S. Araujo, A.F.U. Carvalho, P.M.S. Tabosa, A.C.O. Monteiro-Moreira, M.D.P. Lobo, F.B.M.B. Moreno, B.A.M. Rocha, J.L.S. Lopes, L.M. Beltramini, I.M. Vasconcelos, A trypsin inhibitor purified from Cassia leiandra seeds has insecticidal activity against Aedes aegypti, Process Biochem. 57 (2017) 228–238. doi:10.1016/j.procbio.2017.03.015. [204] S. Banerjee, A.P. Giri, V.S. Gupta, S.K. Dutta, Structure-function relationship of a bio-pesticidal trypsin/chymotrypsin inhibitor from winged bean, Int. J. Biol. Macromol. 96 (2017) 532–537. doi:10.1016/j.ijbiomac.2016.12.018. [205] M.L. Rodrigues Macedo, M.D.G.M. Freire, E.C. Cabrini, M.H. Toyama, J.C. Novello, S. Marangoni, A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae), Biochim. Biophys. Acta - Gen. Subj. 1621 (2003) 170–182. doi:10.1016/S0304-4165(03)000552. [206] P. Lin, T.B. Ng, A stable trypsin inhibitor from Chinese dull black soybeans with potentially exploitable activities, Process Biochem. 43 (2008) 992– 57

Jo

ur na

lP

re

-p

ro

of

998. doi:10.1016/j.procbio.2008.05.005. [207] R. Oddepally, G. Sriram, L. Guruprasad, Purification and characterization of a stable Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds, Phytochemistry. 96 (2013) 26–36. doi:10.1016/j.phytochem.2013.09.010. [208] J. Ruan, J. Yan, H. Chen, C. Jianping, W. Sun, G. Zhao, Purification and properties of the chymotrypsin inhibitor from wild emmer wheat (Triticum dicoccoides) of Israel and its toxic effect on beet armyworm, Spodoptera exigua, Pestic. Biochem. Physiol. 142 (2017) 141–147. doi:10.1016/j.pestbp.2017.06.013. [209] D.D. d. Souza, R.M.P. Brandão-Costa, W.W.C. Albuquerque, A.L.F. Porto, Partial purification and characterization of a trypsin inhibitor isolated from Adenanthera pavonina L. seeds, South African J. Bot. 104 (2016) 30–34. doi:10.1016/j.sajb.2015.11.008. [210] A.C.B. Cruz, F.S. Massena, L. Migliolo, L.L.P. Macedo, N.K.V. Monteiro, A.S. Oliveira, F.P. Macedo, A.F. Uchoa, M.F. Grossi de Sá, I.M. Vasconcelos, A.M. Murad, O.L. Franco, E.A. Santos, Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds, Plant Physiol. Biochem. 70 (2013) 61–68. doi:10.1016/j.plaphy.2013.04.023. [211] E.R. Prasad, A. Dutta-Gupta, K. Padmasree, Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo), Phytochemistry. 71 (2010) 363–372. doi:10.1016/j.phytochem.2009.11.006. [212] L.A. Calderon, H.A. Almeida Filho, R.C.L. Teles, F.J. Medrano, C. Bloch, M.M. Santoro, S.M. Freitas, Purification and structural stability of a trypsin inhibitor from Amazon inga cylindrica [Vell.] Mart. seeds, Brazilian J. Plant Physiol. 22 (2010) 73–79. doi:10.1590/S1677-04202010000200001. [213] S.F.F. Ribeiro, K.V.S. Fernandes, I.S. Santos, G.B. Taveira, A.O. Carvalho, J.L.S. Lopes, L.M. Beltramini, R. Rodrigues, I.M. Vasconcelos, M. Da Cunha, G.A. SouzaFilho, V.M. Gomes, New small proteinase inhibitors from Capsicum annuum seeds: Characterization, 58

Jo

ur na

lP

re

-p

ro

of

stability, spectroscopic analysis and a cDNA cloning, Biopolymers. 100 (2013) 132–140. doi:10.1002/bip.22172. [214] F.P.S. Rufino, V.M.A. Pedroso, J.N. Araujo, A.F.J. França, L.M.A. Rabêlo, L. Migliolo, S. Kiyota, E.A. Santos, O.L. Franco, A.S. Oliveira, Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests’ digestive proteinases, Plant Physiol. Biochem. 63 (2013) 70–76. doi:10.1016/j.plaphy.2012.11.013. [215] S.S. Mohanraj, S.D. Tetali, N. Mallikarjuna, A. Dutta-Gupta, K. Padmasree, Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata, Phytochemistry. 151 (2018) 78–90. doi:10.1016/j.phytochem.2018.02.009. [216] M. Dantzger, I.M. Vasconcelos, V. Scorsato, R. Aparicio, S. Marangoni, M.L.R. Macedo, Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential, Phytochemistry. 118 (2015) 224–235. doi:10.1016/j.phytochem.2015.08.013. [217] P.M.G. Paiva, M.L.V. Oliva, H. Fritz, L.C.B.B. Coelho, C.A.M. Sampaio, Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds, Phytochemistry. 67 (2006) 545–552. doi:10.1016/j.phytochem.2005.12.017. [218] E.F. Fang, J.H. Wong, T.B. Ng, Thermostable Kunitz trypsin inhibitor with cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory activities from Korean large black soybeans, J. Biosci. Bioeng. 109 (2010) 211–217. doi:10.1016/j.jbiosc.2009.08.483. [219] M. Indarte, C.M. Lazza, D. Assis, N.O. Caffini, M.A. Juliano, F.X. Avilés, X. Daura, L.M.I. López, S.A. Trejo, A Bowman–Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid, Planta. 245 (2017) 343–353. doi:10.1007/s00425-016-2611-6. [220] M. DESHIMARU, R. HANAMOTO, C. KUSANO, S. 59

Jo

ur na

lP

re

-p

ro

of

YOSHIMI, S. TERADA, Purification and Characterization of Proteinase Inhibitors from Wild Soja ( Glycine soja ) Seeds, Biosci. Biotechnol. Biochem. 66 (2002) 1897– 1903. doi:10.1271/bbb.66.1897. [221] C. Aguirre, S. Valdés-Rodríguez, G. MendozaHernández, A. Rojo-Domínguez, A. Blanco-Labra, A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis suaveolens L.) inhibits proteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae), Comp. Biochem. Physiol. - B Biochem. Mol. Biol. 138 (2004) 81–89. doi:10.1016/j.cbpc.2004.02.011. [222] S. Klomklao, S. Benjakul, H. Kishimura, M. Chaijan, Extraction, purification and properties of trypsin inhibitor from Thai mung bean (Vigna radiata (L.) R. Wilczek), Food Chem. 129 (2011) 1348–1354. doi:10.1016/j.foodchem.2011.05.029. [223] S.R. Babu, B. Subrahmanyam, Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner), Pestic. Biochem. Physiol. 98 (2010) 349–358. doi:10.1016/j.pestbp.2010.07.008. [224] R.C.L. Teles, E.M.T. De Souza, L.D.A. Calderon, S.M. De Freitas, Purification and pH stability characterization of a chymotrypsin inhibitor from Schizolobium parahyba seeds, Phytochemistry. 65 (2004) 793–799. doi:10.1016/j.phytochem.2004.02.002. [225] I.F. Batista, M.L. Oliva, M.S. Araujo, M.U. Sampaio, M. Richardson, H. Fritz, C. a Sampaio, Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds., Phytochemistry. 41 (1996) 1017–1022. doi:10.1016/00319422(95)00710-5. [226] A.O. El-latif, Isolation and purification of a papain inhibitor from Egyptian genotypes of barley seeds and its in vitro and in vivo effects on the cowpea bruchid, Callosobruchus maculatus (F.), Pestic. Biochem. Physiol. 118 (2015) 26–32. doi:10.1016/j.pestbp.2014.10.016. [227] C. Lazza, S. Trejo, W. Obregon, L. Pistaccio, N. 60

Jo

ur na

lP

re

-p

ro

of

Caffini, L. Lopez, A Novel Trypsin and αChymotrypsin Inhibitor from Maclura pomifera Seeds, Lett. Drug Des. Discov. 7 (2010) 244–249. doi:10.2174/157018010790945832. [228] N. Jackson, L. Czaplewski, L.J. V Piddock, Discovery and development of new antibacterial drugs: learning from experience?, J. Antimicrob. Chemother. 73 (2018) 1452–1459. doi:10.1093/jac/dky019. [229] K. Fosgerau, T. Hoffmann, Peptide therapeutics: current status and future directions., Drug Discov. Today. 20 (2015) 122–128. doi:10.1016/j.drudis.2014.10.003. [230] X. de Lamballerie, P. Colson, The battle against infectious diseases in developing countries: the inseparable twins of diagnosis and therapy., Clin. Chem. 52 (2006) 1217. doi:10.1373/clinchem.2006.071316. [231] D.B. Johnson, A. Schippers, Recent Advances in Acidophile Microbiology: Fundamentals and Applications, Frontiers Media SA, 2017. https://books.google.com.ar/books?id=ZFokDwAAQBA J. [232] E.K. Ulleberg, I. Comi, H. Holm, E.B. Herud, M. Jacobsen, G.E. Vegarud, Human Gastrointestinal Juices Intended for Use in In Vitro Digestion Models., Food Dig. 2 (2011) 52–61. doi:10.1007/s13228-011-0015-4. [233] L. Preiss, D.B. Hicks, S. Suzuki, T. Meier, T.A. Krulwich, Alkaliphilic Bacteria with Impact on Industrial Applications, Concepts of Early Life Forms, and Bioenergetics of ATP Synthesis, Front. Bioeng. Biotechnol. 3 (2015) 75. doi:10.3389/fbioe.2015.00075. [234] L. Terra, P.J. Dyson, M.D. Hitchings, L. Thomas, A. Abdelhameed, I.M. Banat, S.A. Gazze, D. Vujaklija, P.D. Facey, L.W. Francis, G.A. Quinn, A Novel Alkaliphilic Streptomyces Inhibits ESKAPE Pathogens, Front. Microbiol. 9 (2018) 2458. doi:10.3389/fmicb.2018.02458. [235] R.T. Magari, I. Munoz-Antoni, J. Baker, D.J. Flagler, Determining Shelf Life by Comparing Degradations at Elevated Temperatures, J. Clin. Lab. 61

Jo

ur na

lP

re

-p

ro

of

Anal. 18 (2004) 159–164. doi:10.1002/jcla.20016. [236] R.Z. Cer, U. Mudunuri, R. Stephens, F.J. Lebeda, IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding., Nucleic Acids Res. 37 (2009) W441-5. doi:10.1093/nar/gkp253. [237] E. Cunningham, What is a raw foods diet and are there any risks or benefits associated with it?, J. Acad. Nutr. Diet. 104 (2004) 1623. doi:10.1016/j.jada.2004.08.016. [238] D. Treggiari, G. Zoccatelli, R. Chignola, B. Molesini, P. Minuz, T. Pandolfini, Tomato cystine-knot miniproteins possessing anti-angiogenic activity exhibit in vitro gastrointestinal stability, intestinal absorption and resistance to food industrial processing, Food Chem. 221 (2017) 1346–1353. doi:10.1016/j.foodchem.2016.11.020. [239] J.N. Losso, The biochemical and functional food properties of the bowman-birk inhibitor., Crit. Rev. Food Sci. Nutr. 48 (2008) 94–118. doi:10.1080/10408390601177589. [240] A.G. Atanasov, B. Waltenberger, E.M. PferschyWenzig, T. Linder, C. Wawrosch, P. Uhrin, V. Temml, L. Wang, S. Schwaiger, E.H. Heiss, J.M. Rollinger, D. Schuster, J.M. Breuss, V. Bochkov, M.D. Mihovilovic, B. Kopp, R. Bauer, V.M. Dirsch, H. Stuppner, Discovery and resupply of pharmacologically active plantderived natural products: A review, Biotechnol. Adv. 33 (2015) 1582–1614. doi:10.1016/j.biotechadv.2015.08.001. [241] C.R. Singh, Review on Problems and its Remedy in Plant Tissue Culture, Asian J. Biol. Sci. 11 (2018) 165–172. doi:10.3923/ajbs.2018.165.172. [242] B. Miralpeix, H. Rischer, S.T. Hakkinen, A. Ritala, T. Seppanen-Laakso, K.-M. Oksman-Caldentey, T.C. and P. Christou, Metabolic Engineering of Plant Secondary Products: Which Way Forward?, Curr. Pharm. Des. 19 (2013) 5622–5639. doi:http://dx.doi.org/10.2174/138161281131931001 6.

62

Jo

ur na

lP

re

-p

ro

of

LEGENDS TO THE FIGURES Fig. 1: Analysis of plant PPIs stable to temperature. (Panel A) Number of reports per year. In the graph, the number of reports citing PPIs stable (blue bar color) or hyperstable (red bar color) at high temperatures is plotted on the ordinate for each year indicated on the abscissa. (Panel B) Percent of plant PPI types according to their classification. In the graph, the percent of the inhibitors that are stable (blue graph color) or hyperstable (red bar color) are plotted on the ordinate for each inhibitor category indicated on the abscissa. Key to inhibitor type: MCPI: metallocarboxipeptidase inhibitor, CPI: cysteine-protease inhibitor, SPI: serine-protease inhibitor, KI: Kunitz inhibitor, BBI: Bowman-Birk inhibitor.

Fig. 2: Analysis of plant PPIs stable to pH. (Panel A) Number of reports per year. In the figure, the number of reports cited annually in the literature of PPIs stable (blue bar color) or hyperstable (red bar color) to shifts in pH is plotted on the ordinate for each of the years indicated on the abscissa. (Panel B) Percent of PPI pH-stability type. In the figure, the percent of PPIs stable (blue bar color) or hyperstable (red bar color) to shifts in pH are 63

Jo

ur na

lP

re

-p

ro

of

plotted on the ordinate for each of the PPI groups denoted on the abscissa. Key to the PPI group: MCPI: metallocarboxipeptidase inhibitor, CPI: cysteine-protease inhibitor, SPI: serine-protease inhibitor, KI: Kunitz inhibitor, BBI: Bowman-Birk inhibitor.

64

Jo

ur na

lP

re

-p

ro

of

Table 1: Criteria for classifying inhibitors according to the temperature stability Group name Classification criteria 1. Plant PPIs stable to extreme temperatures 1.1 Plant PPIs with high thermostability The inhibitors maintain 25-50% of the 1.1.1 Group IA inhibitory activity at temperatures over 90 °C after a 5-30 min incubation The inhibitors maintain 50-75% of the 1.1.2 Group IB inhibitory activity at temperatures over 90 °C after a 5-30 min incubation The inhibitors maintain above 75% of 1.1.3 Group IC the inhibitory activity at temperatures over 90 °C after a 5-30 min incubation 1.2 Plant PPIs hyper stable to temperatures The inhibitory activity at temperatures 1.2.1 Group IIA over 90 °C remains stable after a 30 min incubation The inhibitory activity at temperatures 1.2.2 Group IIB over 90 °C remains stable after a 60 min incubation The inhibitory activity at temperatures 1.2.3 Group IIC over 90 °C remains stable after a 120 min incubation 2. Plant PPIs stable to extreme pHs The inhibitors maintain 50-90% of the 2.1 Plant PPIs with high inhibitory activity after incubation at pH stability to extreme pHs 3-4 and 10-11 The inhibitors maintain over 70% of the 2.2 Plant PPIs hyper stable to inhibitory activity after incubation at pH extreme pHs 2 and 12

65

of

lP

re

-p

ro

Table 2: Plant PPIs highly stable with respect to temperature Ki: dissociation constant, MW: molecular weight, IA: inhibitory activity, T: trypsin, C: chymotrypsin, SPI: serine-protease inhibitor, MCPI: metallocarboxypeptidase inhibitor, N/D: not determined Protease Ki MW Temperature Incubation R Inhibitor name/s Classification Plant name Origin inhibited (M) (kDa) range (°C) time (min) I 2.46 x 1010 Archidendron ellipticum Archidendron Trypsin and (T) Kunitz Serine protease inhibitor Seeds 20 25-95 60 5 Trypsin Inhibitor ellipticum Chymotrypsin 0.5 (AeTI) x 1010

rn a

(C)

Jo u

Papaya Proteinase Kunitz Inhibitor (PPI)

Caesalpinia bonduc Trypsin Kunitz Inhibitor (CbTI)

Serine protease inhibitor Carica Papaya Seeds Trypsin

Caesalpinia Serine protease inhibitor bonduc

66

Trypsin and Seeds Chymotrypsin

3x 24 10-7

30-100

10

8 7 9 1

60

7 2 9

2.75 x 1010

(T) 0.95 x

20

5-95

of Moringa oleifera Kunitz Protease Inhibitor

Serine protease inhibitor

Cicer arietinum L.

lP

Entada Serine protease inhibitor acaciifolia (Benth.)

Cajanus cajan cv. ICP 7118 BowmanProteinase Birk Inhibitor (C11PI)

Serine protease inhibitor

Sorghum Protease Other SPI Inhibitor

Sorghum Serine protease inhibitor bicolor var. Giza 10

Jo u

(C)

Seeds Trypsin

N/D 30

40-100

Leaves Trypsin

1.5 x 23.6 30-70 10-9

re

Moringa oleifera

10

Solanum Metallocarboxypeptidase Carboxypeptidase tuberosum sv Tubers N/D 4,218 80 inhibitor A Desirée

rn a

Potato Carboxypeptidase MCPI type Inhibitor (PCI) Entada acaciifolia Kunitz Trypsin Inhibitor (EATI)

ro

Serine protease inhibitor

-p

Chickpea Trypsin Other SPI Inhibitor

10-

67

Seeds Trypsin

Cajanus cajan Trypsin and Seeds cv. ICP 7118 Chymotrypsin

Trypsin and Seeds Chymotrypsin

1.75 x 20 10-9

37-100

2.72 x 10-7 (T) 8.38 20-80 3.72 x 10-6 (C) 1.22 x 15.2 20-100 10-9

20 30

1 3 5 5 7 1

15

1

30

8 1 9

30

8 8

45

8 5 1

of ro -p

Zea maize var. Trypsin and Seeds Hi Teck 2031 Chymotrypsin

re

Other SPI

Serine protease inhibitor

lP

Maize Protease Inhibitor

Glycine max Serine protease inhibitor var. Giza 22

Poincianella pyramidalis Kunitz Trypsin Inhibitor (PpyTI)

Poincianella Serine protease inhibitor pyramidalis

Jo u

rn a

Soybean Serine Other SPI Protease Inhibitor

Albizia amara Protease Inhibitor Other SPI (API)

0

10

(T) 20 1.79 x 10-9 (C)

20-100

45

Trypsin and Seeds Chymotrypsin

-

17.9 20-100

45

Seeds Trypsin

1.2 x 19.04 37-100 10-9

30

1.24 x 49 10-8

30

Serine protease inhibitor Albizia amara Seeds Trypsin

68

(T) 1.18 x 10-9 (C) 7.22 x 10-

40-100

8 5 1 0

8 5 1 0 8 7 9 3 1 0 9 3 1

of

Cysteine protease phytocystatin inhibitor

ro

-p

Inga laurina Trypsin Inhibitor Kunitz (ILTI)

Allium sativum Cloves Papain

Serine protease inhibitor Inga laurina

re

Garlic Phytocystatin (GPC)

Seeds Trypsin

0 8.5 x 12.5 30-90 10-8

30

6x 20 10-9

37-100

30

20-100

30

Serine protease inhibitor

Bauhinia variegata var. Kunitz variegata Trypsin Inhibitor (BvvTI)

Bauhinia Serine protease inhibitor variegata var. Seeds Trypsin Variegata

0.1 x 21.09 0-100 10-9

30

Senna tora Kunitz Trypsin Inhibitor

Serine protease inhibitor Senna tora

0.23 x 19.72 30-100 10-9

30

Solanum aculeatissimum

Serine protease inhibitor

1.6 22.2 10-100 x

30

Putranjiva roxburghii

Seeds Trypsin

Jo u BowmanBirk

69

1.4 x 34 1011

rn a

lP

Putranjiva roxburghii Other SPI Trypsin Inhibitor (PRTI)

Seeds Trypsin

Solanum Trypsin and Fruits aculeatissimum Chymotrypsin

2 9 1 7 8 1 4 8 9 9 2 6 ° 8 3 9 1 8 8 9 5 1 4 8 7

re

Butea monosperma

( 9 4 ( 1 2 (

10

(T) 1.45 x 10(C)

Seeds Trypsin

Serine protease inhibitor Allium sativum Bulbs Trypsin

rn a

Allium sativum Protease Inhibitor Kunitz (ASPI)

Serine protease inhibitor

10-

10

lP

Butea monosperma Kunitz Protease Inhibitor (BmPI)

-p

ro

of

Protease Inhibitor (SAPI)

1.2 x 14 10-9 0.8 x 15 10-

10-120

30

37-100

30

10

Serine protease inhibitor Cassia fistula Seeds Trypsin

2.9 x 7 10-9

10-120

30

Cassia leiandra Trypsin Inhibitor Kunitz (ClTI)

Cassia Serine protease inhibitor leiandra

6.25 x 19.48 30-100 10-8

20

Jo u

Cassia fistula Trypsin Inhibitor Kunitz - 1 (CFTI-1)

70

Seeds Trypsin

9 4 1 2 8 8 9 4 1 2 8 8 9 4 1 2 8 7 9

of Rhamnus frangula Protease Kunitz Inhibitor (RfIP1)

Serine protease inhibitor

ro

Serine protease inhibitor

Psophocarpus Trypsin and Seeds tetragonolobus Chymotrypsin

N/D 20

30-100

30

Rhamnus frangula

Leaves Trypsin

rn a

lP

re

-p

Winged bean ChymotrypsinKunitz Trypsin Inhibitor (WBCTI)

N/D 22.4 37-90

30

60

Serine protease inhibitor Cassia grandis Seeds Trypsin

N/D 19

Platypodium elegans Trypsin Kunitz Inhibitor (PeTI)

Platypodium Serine protease inhibitor elegans

Seeds Trypsin

0.16 x 19.7 30-100 10-9

30

Inga edulis

Serine protease inhibitor Inga edulis

Seeds Trypsin

6.2 19.68 30-100

30

Jo u

Cassia grandis Trypsin Inhibitor Kunitz (CgTI)

Kunitz

71

0-100

° 8 6 ( 9 5 ( 1 4 ( 7 7 8 2 9 1 8 8 9 3 1 5 8 7 1 2 8

-p

ro

of

Trypsin Inhibitor (IETI)

Peltophorum dubium Trypsin Kunitz Inhibitor (PDTI)

Serine protease inhibitor

Crotalaria pallida Trypsin Other SPI Inhibitor (CpaTI)

Crotalaria Serine protease inhibitor pallida

Calliandra selloi Trypsin Inhibitor Kunitz (CSTI)

Calliandra Serine protease inhibitor selloi Macbride

Chinese black soybean Trypsin Kunitz Inhibitor (BSKTI)

Serine protease inhibitor

re

Seeds

72

5 9 4 1 3

1.6 x 10Trypsin and Chymotrypsin

lP

rn a

Jo u

Peltophorum dubium

x 10-9

10

(T) 20 2.6 x 10-7 (C)

37-100

30

Seeds Trypsin

N/D 32.5 37-100

30

Seeds Trypsin

2.21 x 20.27 37-100 10-7

30

N/D 20

10

Glycine max Seeds Trypsin cv. Dull Black

0-100

9 8 1 5

8 7 9 ° 5 9 8 8 1 5 9 8 1

of

ro

Serine protease inhibitor Vigna mungo Seeds Trypsin

-p

Black gram BowmanProtease Inhibitor Birk (BgPI)

Trigonella Serine protease inhibitor foenumgraecum

Piptadenia moniliformis Trypsin Kunitz Inhibitor (PmTKI)

Serine protease inhibitor

rn a

Jo u

Kunitz

Piptadenia moniliformis

Trypsin and Chymotrypsin

Seeds

Trypsin and Chymotrypsin

3.01 x 10-9 (T) 19.84 37-100 0.52 x 10-9 (C) 1.5 x 10-8 (T) 3.0 19.29 37-100 x 10-

30

15

9 9 ( 1 7 (

30

9 °

10

(C)

Adenanthera pavonina Trypsin Kunitz Inhibitor

Serine protease inhibitor

Wild Emmer

Serine protease inhibitor Triticum

Other SPI

Seeds

lP

re

Trigonella foenum-graecum Kunitz Trypsin Kunitz Inhibitor (TfgKTI)

3.07 x 8.04 37-100 10-7

7 9 9 1 8

73

Adenanthera pavonina

Seeds Trypsin

N/D 14.1 25-100

30

Seeds Chymotrypsin

2.41 13

30

10-100

8 7 1 6 8

ro

re

-p

Metallocarboxypeptidase Capsicum annuum inhibitor

Jo u

rn a

lP

Yellow Bell Pepper Carboxypeptidase MCPI type Inhibitor (YBPCI)

of

dicoccoides

Chymotrypsin Inhibitor (WeCI)

74

Seeds

x 10-9 (C)

Carboxypeptidase N/D 4.06 100 A

9 9 1 ° 5 min

7

Jo u

rn a

lP

re

-p

ro

of

Table 3: Plant PPIs hyperstable with respect to temperature Ki: dissociation constant, MW: molecular weight, IA: inhibitory activity, T: trypsin, C: chymotrypsin, SPI: serine-protease inhibitor, MCPI: metallocarboxypeptidase inhibitor, N/D: not determined Protease Ki Temperature Inhibitor name/s Classification Plant name Origin MW (kDa) inhibited (M) range (°C) Phaseolus aureus BowmanPhaseolus Trypsin, Serine protease inhibitor Fruit N/D 16.6 30-90 aureus Roxb. Inhibitor Birk Chymotrypsin Cratylia mollis 1.4 8.55 BowmanTrypsin Inhibitor Serine protease inhibitor Cratylia mollis Seeds Trypsin x (CmTI1) 20-100 Birk (CmTI) 10-9 8.62(CmTI2) Korean Bean Trypsin Inhibitor Kunitz Serine protease inhibitor Glycine max Seeds Trypsin N/D 20.10 0-100 (KBTI) Sapindus 2.4 saponaria Sapindus Kunitz Serine protease inhibitor Seeds Trypsin x 18 100 Trypsin Inhibitor saponaria -9 10 (SSTI) 1.4 x 1010 Capsicum Capsicum Trypsin, annuum Trypsin Kunitz Serine protease inhibitor Seeds (T) 6 37-100 annuum Chymotrypsin Inhibitor (CaTI) 4.3 x 10-9 (C) Pithecellobium Kunitz Serine protease inhibitor Pithecellobium Seeds Trypsin 5.7 21 34-100

75

-p

ro BowmanBirk

Clitoria Seeds fairchildiana

Hordeum vulgare

76

10

3.3 x 10Trypsin, Chymotrypsin

(T) 7.97 1.5 x 10-

37-100

10

Seeds

Papain

(C) 1.95 x 12.4-13.9 10-

20-100

12

Maclura Serine protease inhibitor pomifera

Serine protease inhibitor

x 10-

10

re Cysteine protease inhibitor

rn a

BowmanBirk

Jo u

Maclura pomifera Bowman-Birk Inhibitor (MpBBI) Rhynchosia sublobata Bowman-Birk Inhibitor (RsBBI1)

Phytocystatin

Serine protease inhibitor

lP

Clitoria fairchildiana BowmanProteinase Birk Inhibitor (CFPI)

Barley Protease Inhibitor

of

dumosum

dumosum Kunitz Inhibitor (PdKI4)

Rhynchosia sublobata

Seeds

Trypsin

Recombinant Trypsin, (Seeds) Chymotrypsin

6.6 x 6.65 10-8

40-100

3.58 x 10-7 9.97 (T) 4.46

40-100

of ro -p

Seeds

Trypsin

Seeds

Trypsin

N/D 8.7

4-94

Seeds

Trypsin

N/D 14

90

re

Serine protease inhibitor Glycine max

lP

Wild Soja Trypsin inhibitor BowmanI (WSTI-I) and Birk IV (WSTI-IV)

Jo u

rn a

Hyptis suaveolens BowmanHyptis Trypsin Inhibitor Serine protease inhibitor suaveolens L. Birk (HSTI) Trypsin Inhibitor Vigna radiata from mung bean Other SPI Serine protease inhibitor L. R. Wilczek seeds Vigna unguiculata Cysteine Cysteine protease Vigna Phytocystatin Inhibotor 1 unguiculata inhibitor (VuCys1) and VuCys2 Potato Solanum Metallocarboxypeptidase tuberosum Carboxypeptidase MCPI type inhibitor subsp. Inhibitor from

77

x 10-7 (C) 4.6 x 10-9 (-I) 7.52 (-I) 6.2 7.96 (-IV) x 10-9 (IV)

Recombinant Papain, (Leaves) Chymopapain

N/D

10.7 (1) 21.9 (2)

10.8 Tubers and Carboxypeptidase x 4.309 Recombinant A 10-9

90

100

100

of

-p

ro

Opuntia Serine protease inhibitor streptacantha Seeds Lemaire

re

Jatropha Serine protease inhibitor curcas

BowmanBirk

Seed cake Seed cake

Trypsin

N/D 4.19

30-90

Trypsin

2x 10- 10.25

90

Trypsin

lP

Ricinus Serine protease inhibitor communis

Serine protease inhibitor

Jo u

Luetzelburgia auriculata Bowman-Birk Inhibitor (LzaBBI)

Andigenum cv. Churqueña

rn a

Churqueña (chuPCI) Opuntia streptacantha Other SPI Trypsin Inhibitor 2 (OsTI 2) Jatropha curcas Trypsin Inhibitor Other SPI (JcTI-I) Ricinus communis Trypsin Inhibitor Other SPI (RcTI)

11

1.9 x 14 10-5 8.6 x 1011

Luetzelburgia Seeds auriculata

Trypsin, Chymotrypsin

(T) 17.3 1.2 x 1012

(C)

78

100

98

Jo

ur na

lP

re

-p

ro

of

Table 4: Plant PPIs with high stability at extreme pHs* *For the biochemical characterization of the plant PPIs in this table, cf. Tables 2 and 3. Incubation Residual IA Inhibitor name/s pH range time Reference (%) (min) Hyptis suaveolens Trypsin Inhibitor 3-10.7 60 100 [221] (HSTI) Schizolobium parahyba Chymotrypsin Inhibitor 2-12 10 80 [224] (SPCI) pH 1-4: 75 Phaseolus aureus 1-13 30 pH 12-13: [72] Inhibitor 75 Archidendron ellipticum 1-12 120 pH12: 66 [186] Trypsin Inhibitor (AeTI) Cratylia mollis Trypsin 2-10 30 100 [217] Inhibitor (CmTI) Inga laurina Trypsin 2-10 30 95 [201] Inhibitor (ILTI) Calliandra selloi pH 2-3: 80 Trypsin Inhibitor 2-11 30 pH 8.5-11: [175] (CSTI) 80 pH 3-5: 70 Chickpea Trypsin 2-12 180 pH 2, 11, 12: [194] Inhibitor 5 Opuntia streptacantha Trypsin Inhibitor 2 3.8-9 120 100 [21] (OsTI 2) Bauhinia variegata var. pH 2: 60 variegata Trypsin 2-12 30 [195] pH4-12: 100 Inhibitor (BvvTI) pH 2-3: 20 pH 4-10: 55Moringa oleifera 2-12 180 65 [38] Protease Inhibitor pH 11-12: 30 Trypsin Inhibitor from pH 2-3: 90 2-10 30 [222] mung bean seeds pH 10: 97 Sapindus saponaria 2-10 60 90 [22] Trypsin Inhibitor (SSTI) Entada acaciifolia 2-10 60 100 [184] 79

Sorghum Protease Inhibitor

2-12

180

Maize Protease Inhibitor 2-12

180

Jatropha curcas Trypsin 2-10 inhibitor (JcTI-I)

30

Soybean Serine Protease 2-12 Inhibitor

lP

180

2-10

60

2-10

60

ur na

Clitoria fairchildiana Proteinase Inhibitor (CFPI) Poincianella pyramidalis Trypsin Inhibitor (PpyTI)

Jo

Butea monosperma Protease Inhibitor (BmPI)

of

30

pH 3-5: 80/50-75 (T/C) pH 9: 90/80 [223] (T/C) pH 10: 85/60 (T/C) pH 2-3: 30 pH 11-12: 30-10 pH 2-3: [190] 50/35 (T/C) pH 11: 55/20 (T/C) pH 12: 10 100

[123]

pH 2-3: 5070 pH 4-9: 80 [188] pH 10, 11, 12: 70, 30, 0 pH 2-4: 8595 pH 10: [216] 90/82 (T/C)

re

Trigonella foenumgraecum Kunitz Trypsin 3-10 Inhibitor (TfgKTI)

90/60 (T/C) [213]

ro

30

-p

Trypsin Inhibitor (EATI) Capsicum annuum 2-10 Trypsin Inhibitor (CaTI)

2-12

Solanum aculeatissimum Protease 2-12 Inhibitor (SAPI)

180

60

80

pH 2: 90 [191] pH 3-10: 95 pH 2-3: 1020 pH 11: 40 [139] pH 12: 25 pH 13: 10 pH 2: 20 pH 3-5: 5070 [146] pH 10-12: 68/60-44/32 (T/C)

180

Cassia fistula Trypsin Inhibitor - 1 (CFTI-1)

2-13

180

Allium sativum Protease 1-12 Inhibitor (ASPI)

180

Rhamnus frangula Protease Inhibitor (RfIP1)

180

2-13

Cassia leiandra Trypsin 2-10 Inhibitor (ClTI)

[189]

[199]

[181]

[196]

[203]

30

95

[198]

60

pH 2: 65 pH 12: 80

[25]

180

80/75 (T/C) [26]

60

100

80

pH 2-4: 10 pH 5-6: 20[187] 30 pH 9-10: 20 pH 2-5: 2060 [197] pH 11-12: 70

re

30

2-10

30

lP

Garlic Phytocystatin (GPC)

pH 3-5: 4060 pH 9: 20 pH 2-3: 15 pH 4-5: 70 pH 11-13: 40-10 pH 3-5: 60 pH 9: 80 pH10-12: 70-40 pH 2-4: 3060 pH 5-10: 80 pH 11-13: 60-30

-p

Albizia amara Protease 2-9 Inhibitor (API)

pH 9-11:70 [122]

of

60

ro

Ricinus communis 2-11 Trypsin Inhibitor (RcTI)

Cassia grandis Trypsin 2-12 Inhibitor (CgTI)

ur na

60

Jo

Inga edulis Trypsin 2-10 Inhibitor (IETI) Yellow Bell Pepper Carboxypeptidase 2-12 Inhibitor (YBPCI) Luetzelburgia auriculata Bowman2-11 Birk Inhibitor (LzaBBI) Platypodium elegans 2-10 Trypsin Inhibitor (PeTI)

81

[200]

Jo

ur na

lP

re

-p

ro

of

Table 5: Plant PPIs hyperstable to extreme pHs* *For the biochemical characterization of the PPIs in this table, cf. Tables 2 and 3. Incubation Residual IA Inhibitor name/s pH range Reference time (min) (%) Enterolobium contortisiliquum Trypsin 2-12 10 100 [225] Inhibitor (ECTI) Wild Soja Trypsin Inhibitor I (WSTI-I) and 2-12 60 95 [220] IV (WSTI-IV) Peltophorum dubium 1.5-9.5 60 95 [205] Trypsin Inhibitor (PDTI) Crotalaria pallida Trypsin Inhibitor 2-12 60 90 [180] (CpaTI) Papaya Proteinase 1.5-11 180 95 [192] Inhibitor (PPI) Caesalpinia bonduc 1-12 180 98 [185] Trypsin Inhibitor (CbTI) Putranjiva roxburghii pH 2-5, 102-12 30 [177] Trypsin Inhibitor (PRTI) 12: 95 Chinese black soybean Trypsin Inhibitor 1-14 120 100 [206] (BSKTI) Korean Bean Trypsin pH 2: 90 2-12 30 [218] Inhibitor (KBTI) pH 12: 90 Black gram Protease 2-12 60 95 [211] Inhibitor (BgPI) Piptadenia moniliformis Trypsin Kunitz Inhibitor 2-12 30 100 [210] (PmTKI) Pithecellobium dumosum Kunitz 2-12 30 90 [214] Inhibitor (PdKI-4) Senna tora Trypsin pH 2, 11, 12: 2-12 30 [202] Inhibitor 70 Cajanus cajan cv. ICP 7118 Proteinase 2-12 60 pH 12: 85 [157] Inhibitor (C11PI) pH 2: 80 Barley Protease Inhibitor 2-12 180 [226] pH 12: 85 Maclura pomifera 2-13 30 100 [219] 82

30

98

[24]

30

95

[208]

60

100

[215]

Jo

ur na

lP

re

-p

ro

of

Bowman-Birk Inhibitor (MpBBI) Vigna unguiculata Cysteine Inhibotor 1 2-11 (VuCys1) and VuCys2 Wild Emmer Chymotrypsin Inhibitor 2-12 (WeCI) Rhynchosia sublobata Bowman-Birk Inhibitor 2-12 (RsBBI1)

83

ro

of

Table 6: Plant PPIs with physicochemical stability and biologic activity* *For the biochemical characterization of the PPIs in this table, cf. Tables 2 and 3. Biological Inhibitor´s Description activity name Pesticide activity

Reference

re

-p

Inhibitory activity on larval gut proteases Hyptis Inhibitory activity against trypsin-like suaveolens proteases from Prostephanus [221] Trypsin truncatus and Manduca sexta. Inhibitor (HSTI)

Inhibitor (CbTI)

Opuntia streptacantha

lP

Caesalpinia Inhibitory activity against trypsin-like bonduc Trypsin [185] proteases from Spodoptera litura.

rn a

Inhibitory activity against trypsin-like proteases from Prostephanus

Trypsin Inhibitor 2 (OsTI 2)

truncatus, Periplaneta americana, Acheta sp and Gryllus sp.

Inhibitory activity against trypsin-like proteases from the Anagasta

Jo u

Sapindus saponaria

Trypsin Inhibitor (SSTI)

Senna tora Trypsin

84

[21]

kuehniella, Corcyra cephalonica , [22] Diatreae saccharalis and Anticarsia gemmatallis. Inhibitory activity against Helicoverpa [202] armigera trypsin-like proteases.

of

Inhibitor

ro

Inhibitory activity on larval development Peltophorum Development delay on Anagasta dubium Trypsin kuehniella larvae. Inhibitor (PDTI)

-p

Archidendron ellipticum

lP

re

Mortality against Spodoptera litura. Trypsin Inhibitor (AeTI) Sorghum and Mortality against Spodoptera Maize Protease littoralis. Inhibitor

Poincianella pyramidalis

Decrease in both larval weight and survival of Anagasta kuehniella larvae, besides a larval stage extension.

rn a

Trypsin Inhibitor (PpyTI)

Jo u

Soybean Serine Protease Inhibitor

Barley Protease Inhibitor

85

[205]

[186]

[190]

[191]

Negative effects on the mean larval weight, larval mortality, pupation, [188] and mean pupal weight of Spodoptera

littoralis.

Barley protease inhibitor prolonged the development of Callosobruchus maculatus in proportion to protease inhibitor concentration. Feeding

[226]

of

Callosobruchus maculatus on a diet

rn a

lP

re

-p

ro

containing barley protease inhibitor caused larval mortality ranging from 10.03 ± 2.34 to 29.08 ± 4.02%. Negative effect on mean larval weight, Cassia fistula decline of the fertility and fecundity Trypsin of the moths, and extended total [199] Inhibitor - 1 developmental duration of (CFTI-1) Helicoverpa armigera life cycle. Negative effects on growth and development of Spodoptera exigua. Wild Emmer WeCI significantly increased the Chymotrypsin [208] mortality rate of Spodoptera exigua Inhibitor (WeCI) and caused a significant decrease in its fertility .

Cassia grandis

Trypsin Inhibitor (CgTI)

Mortality against Nasutitermes corniger.

Jo u

Negative effects on Spodoptera Platypodium frugiperda development and weight elegans Trypsin Inhibitor (PeTI)

gain, besides extending the insect life cycle.

[197]

[200]

Inhibitory activity on larval gut proteases and larval development Crotalaria Inhibitory activity against gut [180] pallida Trypsin proteases from Spodoptera

86

frugiperda, Alabama argillacea, Plodia interpunctella, Anthonomus grandis, Zabrotes subfasciatus, Callosobruchus maculatus and Ceratitis capitata.

ro

of

Inhibitor (CpaTI)

-p

Mortality and mass decrease on Callosobruchus maculatus and

Ceratitis capitata.

capitata.

rn a

Kunitz Trypsin Inhibitor (PmKTI)

lP

Piptadenia moniliformis

Inhibitory activity against Helicoverpa armigera both in vivo and in vitro. [194] Decline in larval weight, growth and survival. Inhibitory activity against gut proteases from Anthonomus grandis, Plodia interpuncptella, and Ceratitis [210]

re

Chickpea Trypsin Inhibitor

Jo u

Cajanus cajan cv. ICP 7118 Proteinase Inhibitor (C11PI)

87

Negative effects on Ceratitis capitata larvae development. Inhibitory activity against Achaea janata trypsin-like proteases. Mortality and reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates.

[157]

Clitoria fairchildiana

ro

of

Inhibitory activity against trypsin enzymes from Anagasta kuehniella, Diatraea saccharalis and Heliothis

virescens.

Proteinase Inhibitor (CFPI)

[216]

Jo u

rn a

lP

re

-p

Negative impact on Anagasta kuehniella development . Inhibitory activity on trypsin-like proteases of Helicoverpa armigera. Butea Mortality and reduction in growth monosperma and weight of Helicoverpa armigera. [139] Protease The fertility and fecundity of Inhibitor (BmPI) Helicoverpa armigera, declined whereas the larval–pupal duration of the insect life cycle extended. Winged-Bean Inhibitory activity on Helicoverpa Chymotrypsin- armigera trypsin-like proteases. Trypsin Growth retardation, delayed pupae [204] Inhibitor formation and higher mortality of (WBCTI) Helicoerpa armigera larvae. Inhibitory activity against Aedes aegypti midgut proteases. Cassia leiandra Toxicity on the 3rd instar larvae of Trypsin [203] Aedes Aegypti, 24-h delay of the Inhibitor (ClTI) larvae development and 44% mortality after ten days of exposure.

88

of

Rhynchosia sublobata

Inga edulis

Colletotrichum gloeosporioides.

[122]

Antifungal activity against Candida ssp., including Candida buinensis and [198] Candida tropicalis.

rn a

Trypsin Inhibitor (IETI) Antimicrobial activity

Inhibited the spore germination of

lP

Trypsin Inhibitor (RcTI)

re

Antifungal activity Ricinus communis

[215]

-p

Bowman Birk Inhibitor (RsBBI1) Antifungal and/or antimicrobial activity

ro

Inhibitory activity on Achaea janata trypsin-like proteases. Deleterious effects on larval growth and development in Achaea janata.

Antibacterial activity against Salmonella enterica subspecie Trypsin enterica serovar choleraesuis and Inhibitor (JcTI-I) Staphylococcus aureus.

Jo u

Jatropha curcas

Luetzelburgia auriculata

Antibacterial activity against Staphylococcus aureus.

Bowman-Birk Inhibitor (LzaBBI)

Antifungal and antimicrobial activity

89

[123]

[26]

[189]

[196]

Jo u

rn a

lP

re

-p

ro

of

Antifungal activity against Alternaria alternata, Alternaria tenuissima, and Albizia amara Candida albicans. Protease Antibacterial activity against Inhibitor (API) Pseudomonas aeruginosa and Bacillus subtilis. Inhibitory activity toward commercially trypsin-like proteases Rhamnus from Aspergillus oryzae, Bacillus sp, frangula and Bacillus licheniformis. Protease Antibacterial effect against both Inhibitor (RfIP1) Gram-positive and Gram-negative bacteria. Antiviral and antitumoral activity Chinese Black Inhibitory activity against HIV-1 Soybean reverse transcriptase. Trypsin Anti-proliferative activity forward Inhibitor tumor cells and ability to elicit nitric (BSKTI) oxide production from macrophages. Bauhinia Inhibitory activity against HIV-1 variegata var. reverse transcriptase. variegata Induction of cytokines and apoptotic Trypsin bodies Inhibitor Anti-proliferative activity toward (BvvTI) nasopharyngeal cancer CNE-1 cells.

90

[206]

[195]

lP

re

-p

ro

of

Inhibitory activity against HIV-1 reverse transcriptase. Induction of the the release of proinflammatory cytokines such as TNFα, IL-1β, IL-2 and interferon-γ at the Korean Bean mRNA level. Trypsin [218] Weak antiproliferative activity toward Inhibitor (KBTI) CNE-2 and HNE-2 nasopharyngeal cancer cells, MCF-7 breast cancer cells, and Hep G2 hepatoma cells. KBTI was destitute of mitogenic, ribonuclease and antifungal activities Anticoagulant activity

rn a

Enterolobium contortisiliquum Increase in activated partial Trypsin Inhibitor (ECTI)

Maclura pomifera

thromboplastin time (APTT).

Jo u

Increase inactivated partial thromboplastin time (APTT) but not the thrombin time (TT) nor the prothrombin time (PT).

Bowman-Birk Inhibitor (MpBBI)

91

[225]

[227]