Review on surface modification of nanocarriers to overcome diffusion limitations: An enzyme immobilization aspect

Biochemical Engineering Journal 158 (2020) 107574

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Review on surface modification of nanocarriers to overcome diffusion limitations: An enzyme immobilization aspect

T

Carlin geor malara, Muthulingam Seenuvasanb,*, Kannaiyan Sathish Kumarc,*, Anil Kumard, R Parthibanc a

Department of Biotechnology, Rajalakshmi Engineering College, Thandalam, India Department of Chemical Engineering, Hindusthan College of Engineering and Technology, Coimbatore, India c Department of Chemical Engineering, SSN College of Engineering, Kalavakkam, Tamilnadu, India d Analytical and Environmental Science Division, CSIR-Central Salt & Marine Chemicals Research Institute, Gujarat, India b

H I GH L IG H T S

immobilization is a well-known technique that improve the stability of enzymes. • Enzyme recent, studies on nanomaterials as carriers for immobilization are reported. • AtDiffusion limitations in a reaction is a great barrier for catalytic activity of immobilized enzyme. • Surface modification of nanocarriers have been revealed to be the best option to overcome the diffusional limitations. •

A R T I C LE I N FO

A B S T R A C T

Keywords: Catalytic Biochemical reaction Enzyme Immobilization Diffusion limitations

Immobilized enzymes are widely used in the industries for various biochemical reactions in order to gain the benefits of higher stability and reusability. Emerging trends of nanotechnology in immobilization have extended their major applications as carriers for the enzymes. Nanomaterials exhibit different, unique properties like high surface area to volume ratio, high reactivity, catalytic activity, rigidity, solubility, semi-conductivity and size dependent optical properties. But it is a great challenge for the nanomaterials to exhibit these unique properties completely in the reaction system due to the diffusion limitations. This review focusses on the involvement of diffusion and diffusion limitations in various catalytic reactions, methods to evaluate diffusion limitations and different strategies to overcome. This review can be a different perspective towards the biochemical reactions and can provide information to carry out the enzyme substrate reaction processes using nanomaterials as carriers overcoming diffusion limitations.

1. Introduction

onto nanocarriers and the available strategies to overcome it with a special reference to surface behaviors and modification of carriers.

Inert materials are used as carriers for immobilizing enzymes effectively for easy recovery, potential continuous use, withstanding capability over extended temperature and pH, economical feasibility, less product contamination and improved stability and activity over the free enzymes. Many industries utilize such advantages for improving the properties like stability, specificity and firmness of the biocatalysts using the nanomaterials because of their excellent properties. However, in addition to the above said advantages, the immobilized enzymes are ought to overcome various diffusion limitations imposed by substrate and the product for an effective biochemical reaction. This review focusses on the effect of diffusion limitations on the enzyme immobilized

⁎

1.1. Catalytic reactions using enzymes Enzymes are usually the secondary metabolites of the microbial sources and can be an intracellular or extracellular product of any biological substance. Isolation and storage of such enzymes in their free-state is an economically challenging task concerning the stability that hinder their extended potential industrial use. Hence, in order to improve certain tunable properties of the enzymes, including stability, specificity, catalytic activity, and affinity towards the substrate, the biotechnological techniques are involved. These ameliorated

Corresponding authors. E-mail addresses: [email protected] (M. Seenuvasan), [email protected] (K.S. Kumar).

https://doi.org/10.1016/j.bej.2020.107574 Received 18 December 2019; Received in revised form 23 March 2020; Accepted 24 March 2020 Available online 29 March 2020 1369-703X/ © 2020 Elsevier B.V. All rights reserved.

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Table 1 Various enzymes and their industrial applications. Sl. No.

Industrial Sector

Applications (Purpose)

Enzyme

References

1

Agriculture

2

Food

3 4

Paper & Pulp Textile

5

Pharmaceutical

Enzyme assisted silage fermentation Production of animal feed supplements Processing of crops Pesticides Meat tenderization Cheese making Brewing/Baking Beer production Juice clarification Coating (by reducing viscosity of starch) Warp sizing Bleaching Bioscouring Bio polishing Killing disease causing microorganisms Wound healing Diagnosis Biosensor

[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]

6 7

Energy Water Treatment

Amylase, Amyloglycosidase, Cellulase, Hemicellulase Phytases Glucoamylase, α-amylase & glucose, isomerase Protease, phosphotatease Protease Lipase Amylase Glucoamylase Pectinase, Xylanase Amylase Amylase Laccase Cellulase, Lipase Cellulase Protease Peroxidases, protease Urease Alkaline phosphatase Glucose Oxidase Lipase Peroxidase Lignolytic enzymes Lignolytic enzymes

Biofuel production Phenol removal Heavy metal removal Waste degradation

[19] [20] [21] [22]

1.3. Influencing factors of biochemical activity in immobilized enzyme

characteristics of enzymes extend their biochemical applications in various sectors including agriculture, food, paper & pulp, textile and pharmaceutical industries. The industrial applications of each enzymes varies based on the specific activity and are used for various purposes as listed in Table 1. Enzymes are well known as highly efficient catalysts because of their ability to accelerate the reactions [23–25]. However, the factors enhance the reaction rates are still unclear [26,27]. “Lock-and-key” and “induced-fit” hypotheses explain the activity of enzymes by the interactions with substrate corresponding to the structure of enzymes [28,29].

During immobilization of enzyme onto the carriers, certain unavoidable chances occur in the active sites to remain concealed from the surface that may offer negative impact on the activity [33]. For a prominent catalytic reaction, it is necessary for the substrate to reach the active site of the enzyme by diffusion through the reaction medium. At the same time, the products should diffuse away from the active site, assisting further binding of substrate. 1.3.1. Immobilization methods The methods of attaching the enzyme onto the carriers play an important role in the performance of the immobilized enzyme. Various methods of immobilization include entrapment [38], covalent binding [39,40], encapsulation, and adsorption [41]. Weaker forces are involved in the adsorption method and hence surface area of the carrier impose an effect on the enzyme loading. Immobilization through chemical bonds in covalent binding method deliver excellent tolerance ability towards varying conditions and improved stability.

1.2. Immobilization of enzymes Immobilized enzymes are those that are physically localized and confined in a certain defined region of space retaining their catalytic activity. Also, it is one of the most commonly used technique to maintain the stability of the enzyme that tends to be lost in the native state. Immobilization makes recovery and potential reuse of the enzymes easy and make the catalytic processes cost effective [30,31]. Several other advantages of immobilization include multi-enzyme reaction system, easy separation of the enzyme, enzyme removal from the system at any point of reaction, prevention of enzyme loss and enhanced catalysis. Genetic modifications are aimed to enhance the activity of the enzymes by altering the binding chemistry [32] and the site-directed mutagenesis is involved in controlling the orientation of the enzyme to improve its catalytic activity. In spite of these specific alterations through genetic modification, chemical re-engineering [33] and site-directed mutagenesis [34,35], immobilization is considered to be a successful method. This is because of the advantages of easy separation, possibility of continuous reactions and increased enzyme stability. Different types of immobilization techniques can be used based on the mechanism such as covalent binding, entrapment, encapsulation, adsorption, ionic bonding, affinity binding and metal ion binding as shown in Fig. 1. In spite of numerous types of immobilization, its success rate relies on certain factors like maintaining the catalytic property, stability, recyclability, easy separation, active site orientation and mass transfer. Additionally, every method involves the diffusion limitations in their own way depending on the inorganic and insoluble organic carriers used in immobilization [36,37].

1.3.2. Concentration gradient Concentration gradient is one of the important factors that serve as the driving force for the net diffusive process which is the movement of solute from higher to lower concentration. The increased concentration gradient eases the diffusion process leading to increased reaction rate by making the substrate molecule readily available for the enzyme [35]. When the level of substrate attains saturation, diffusion is slowed down such that the activity of enzyme decreases. 1.3.3. Size of the carrier Secondly, the size of the carrier plays a major role in determining the activity of the immobilized enzyme [73]. This is because of the inverse relation between the size of the carrier and the enzyme loading. Reduction in size of the carrier avails higher surface area for enzyme binding. 1.3.4. Surface porosity of the carrier Surface porosity of the carriers is one of the most important influencing factor of the activity of immobilized enzyme. It is believed that highly porous carriers provide numerous binding sites for the enzyme molecules than the non-porous carriers. In spite of high enzyme 2

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Fig. 1. Various types of immobilization based on the mechanism.

loadings, very few active sites are disclosed for the substrates. This makes the porous supports less efficient to be used as a carrier for enzyme immobilization. Another cause is the demand for the reactants to diffuse through the pores of the carrier making the catalysis complex. The diameter of the pores on the surface and the molecular velocity governs the Knudsen diffusion. In the event of non-porous carriers, enzyme loading and the adherence of enzyme remains less, but the resulting activity of the immobilized enzyme is noticeable.

The most important factor to be considered influencing the activity of immobilized enzyme is the surface porosity of the carriers. The catalytic reactions become complex with the need of diffusion of reactants through the surface pores. Knudsen diffusion is the ultimate contributor that is governed by molecular velocity and diameter of the pore on the surface of catalytic carrier.

1.3.5. Diffusion For an effective immobilized enzyme to catalyze the reaction, the substrates should diffuse the reaction medium in order to reach the active site of the immobilized enzyme. Also, the products should diffuse away from the active site, facilitating further binding of substrate. One of the major factors that serve as the driving force for the net diffusive process is concentration gradient ie, movement of solute in the direction of higher to lower concentration. Hence it is necessary for the substrate to cross both the internal and external diffusion. This is because, the surface of the enzyme has an unstirred thin layer of solution termed as ‘Nernst layer’ and when substrate encounter the active enzyme, it tries to diffuse through this layer to reach the active site. External and internal diffusion can be described based on the movement of the solutes in the immobilized enzyme.

In addition to the above discussed factors, diffusion limitations are one of the most significant parameters to be considered to utilize the catalytic activity of immobilized enzyme effectively. Importantly limitations of mass transfer by diffusion are to be of concern for heterogeneous and homogeneous catalysis. Generally, in catalytic reactors like photocatalytic reactors, when mass transport is not fast enough to keep up with catalysis, the overall reaction rate changes. In a recent study by Motegh et al., [42] photoninduced diffusion limitations were assessed in photocatalytic reactors and a study by Nikoshvilli et al. [43], reported about stirring in the catalytic reactors to avoid external and internal diffusion limitations.

1.4. Diffusion limitations

1.5. Recent techniques to overcome diffusion limitations Table 2 gives some significant strategies employed previously to overcome the drawback of diffusion limitations in an immobilized enzyme. Altering the behavior of the carriers for immobilizing enzyme seems to be one of the major strategies. Various carrier molecules are used for immobilization of the enzyme with various conditions for effective retrieval after performing biochemical reactions. With consideration to all process parameters and process variables, non-

• External diffusion occurs in series with the catalytic conversion of •

substrates to products with the inward and outward movement of substrates and products respectively. Internal diffusion occurs in parallel with the movement of substrate and product within the pores of the immobilized enzyme.

3

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Table 2 Strategies and the mechanisms involved in overcoming diffusion limitations. Strategy Used

Mechanism involved

References

Thermally reversible hydrogels as carrier pH-Sensitive Soluble-Insoluble matrices as carrier

Thermal cycling enhances mass transfer of substrate into and of product out of the gel. Cellulase immobilized to a meth acrylic acid/methyl methacrylate, that is soluble above pH 5.0 and insoluble below pH 3.9. Amylase immobilized onto thermos-responsive polymer. Under pressure cycling operation, diffusive flow and convective flow achieved. Thermo dialysis process brings the substrate in and moves the product out. Physical adsorption and covalent binding makes enzyme readily available for the substrate. Avoids the traffic jam of substrate-products inside the carrier.

[44] [45]

Temperature-sensitive Soluble-Insoluble matrices as carrier Pressure-sensitive gels as carrier Reaction under non-isothermal conditions Immobilization of enzymes on carriers’ surface Non-porous carriers

[46] [47] [48] [49] [50,51,52,53,54,55,56]

nanosystem such as macro systems and microsystems seem to be less effective than the nanosystem. The studies using thermally reversible hydrogels and carriers, enhanced the mass transfer of the substrate into and out of the gel [44,46]. The immobilization of enzymes on the surface of the carrier enabled the ready availability if the substrate [49] whereas the attachment onto non-porous carrier surface [50] eliminated the crowding of the substrate molecules in the vicinity of carrier.

fuses to form a single nanoparticle during which diffusion of the nuclei overcome the diffusion limitations occurring with an effort across an imaginary diffusive layer. Fan et al. [72] performed a distinct methodology for synthesizing nanotubes and hollow nanoparticles that involved kirkendall effect ie., supersaturation by different diffusivities. This undesirable effect was avoided by an efficient coalescing of the voids into a hollow core forming hollow nanostructures.

1.6. Nanomaterials as versatile carrier

2.2. In surface modification of nanoparticles

Intervention of nanotechnology in recent years has gained its attention as nanocarriers for enzyme immobilization. This is because of the unique properties (as listed in the Table 3) of nanomaterials like large specific surface area to volume ratio [63], increased stress resistance [64] and decreased mass transfer resistance. Nanomaterials are available in different forms like matrices [65], branched dendrimers [66], nanoparticles [67], fibres [68], tubes [69] and dots [70] to enable enzyme immobilization easier. It can be clearly understood from Table 3 that apart from certain common properties, each nanoparticles has their own unique properties and serve as best in their own way. Hence nanomaterials are chosen based on their application and also their commercial aspects [71].

Diffusion limitations are noticeable during surface modification of nanoparticles due to the presence of interactions between each grafted molecule and the nanoparticle surface. Restricted behaviors of nanomaterials in different solvents limits their applications and hence surface modification is an important strategy that tune the properties of nanomaterials to extend the applications. The surface modification can either alter the existing property or can introduce completely newer property onto nanoparticles using various agents like organosiloxanes, EDC, carbodiimide, amino silanes like APTES [40], AEAPS [73] and silica. Therefore, surface functionalization by coating attributes improvement in the processes and the surface coating that can mask the undesirable characteristics of nanoparticles and that supports further attachments are highly desired in biological applications. Zhang et al [74] reported a reliable recovery of cyclodextrin glycosyltransferase through the iron oxide nanoparticles surface modified using Polydopamine. The diffusion limitations were seen to be reduced after surface modification through higher stability. Cao et al [75] studied the enhancement of oxidase activity on the selenium nanoparticles surface modified by chitosan. Greater improvement was noticed after the addition of stabilizer.

2. Diffusion limitations in a nanosystem This section details about diffusion limitations occurring at various stages in a nanosystem such as during their synthesis, surface modification, reaction stage, attachment with other molecules and in polymer matrix. 2.1. In synthesis of nanoparticles

2.3. In suspension of nanoparticles Generally nanoparticles are synthesized either by ‘Top-down’ or by ‘Bottom-up’ approach. In the classical growth of the nanoparticles, two important mechanisms are involved, such as surface reaction and the diffusion of monomers to the surface (Fig. 2). Numerous small nuclei

Understanding the diffusional behaviors of nanoparticles under suspension is very helpful to overcome limitations easily. Recently, Rudyak et al [76] have well reported the transport process of

Table 3 Nanoparticles and their unique properties. S. No Nanoparticles Unique Properties Enzymes used References Metallic Nanoparticles - Size dependent optical behaviors, high surface area to volume ratio, high reactivity, high stability and chemical inertness. Tunable morphology, spatial confinement, elastic and high strength. 1 2 3

Iron oxide Silver Gold

Superparamagnetic, non-toxic, biocompatible High electrical conductivity, low sintering temperatures Optoelectronic behavior, anti-cancer, biocompatible, quench fluorescence 4 Selenium Anti-cancer, immunomodulation 5 Titanium dioxide Photocatalytic, UV-resistant, degrading organic contaminants Non-metallic Nanoparticles - Polymeric in nature and are readily available. 6 Silica Thermal stability, low toxicity, biocompatible 7 Chitosan Biodegradable, biocompatible, antifungal

4

Pectinase, urease, amylase Lipase, amylase, glucose oxidase Urease, laccase, Lipase

[37] [57] [58]

Chymotrypsin Cellulase, laccase

[59] [60]

Lipase Proteinase, galactosidase

[61] [62]

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Fig. 2. Growth of Nanoparticles.

nanoparticles in fluids and the attraction between nanoparticles and fluid components. Two phases can be considered under fluid systems such as gas-solid and liquid-solid, with a note that solid refers nanoparticles. Presence of a diffusive layer between the fluid molecule and the nanoparticle restricts the easy dispersion of the nanoparticles. The reaction of the nanoparticles with fluid are also mainly limited by the attractions with the fluid components and the molecular size of the surrounding fluid [77].

assumed to be composed of numerous grains and diffusion of isotopic tracer through grains is followed and evaluated numerically [84]. This method measures the diffusion coefficients rapidly as a result of faster diffusion of the tracer through the grains. Also, it is limited for less density, porous samples and the nanocrystals in which diffusion is induced by grain boundary migration.

2.4. In attachment with nanoparticles

NMR is mainly used to study the diffusion in the metal nanoparticles. The NMR spectra details the surface diffusion between nanoparticles and adsorbed gases and also the shape and width of lines in NMR spectra can be used to discriminate the bulk and nanoparticle interface. The hydrodynamic size of the nanoparticles can also be measured using NMR signals that perform particle diffusion similar to the dynamic light scatting technique [85]. Here, the diffusion coefficients are determined using the Brownian movement of the nanoparticles in a dispersed medium and the spin-lattice relaxation time with relation to the decay of magnetization is generally used to determine the diffusion coefficients. Therefore, in general, both slow and fast diffusion in the metal nanoparticles can be estimated from the NMR spectra.

3.2. Nuclear magnetic resonance (NMR) techniques

Recent studies have reported on the attachment of nanoparticles on various substrates like polymer [78], proteins [79] and membrane receptors [80]. The distance dependence limits the attachment locally. But when the nanoparticles are attached to the live cell membranes, the surface of nanoparticles is modified in such a way that the diffusion limitations are eliminated [81]. 2.5. Diffusion of nanoparticles in polymer matrix Solutions, melts and gels are considered to be composed of the polymer matrices and the nanoparticles are entrapped in the polymer matrix for numerous applications including drug delivery [66], sensors, and adsorbent. Here, the nanoparticles have to overcome various free energy barriers among the adjacent molecules. Cai et al [82] reported the topology of the matrix to be a network of untangled and tangled solids such that the nanoparticles perform hopping diffusion depending on the mesh size. Similarly, sub diffusive motion of nanoparticles were reported by Omari et al [83] in semi dilute polymer solutions.

3.3. Conductivity measurements This is one of the simplest and easiest method to determine the diffusion coefficient. Here, the relations between diffusion coefficient and conductivity as employed to evaluate the diffusion in nanosystem. Also, this method is primarily used in the catalytic processes where the nanoparticles are either used as catalyst or as a carrier to attach the catalyst. Comparing the conductivity at various stages of the process helps in estimating the diffusion coefficients [86].

3. Techniques to evaluate diffusion in nanosystem It is very essential to determine the diffusion continuously to confirm the efficiency of the reactions. The importance and role of diffusion in nanosystem are clearly understood from the previous section. Diffusion through the boundary layer of the nanocrystalline solids can be determined using various experiments. In specific, these experiments with relation to the diffusion and the porous nature of nanomaterials.

4. Surface modification of nanocarriers to overcome diffusion limitations The impact of surface porosity on efficient activity of immobilization is discussed in previous sections. Hence it is clear that the surface porosity alteration (or) surface modification of the nanocarriers is significant. In order to modify the surface of the nano carriers, it is essential to investigate the peripheral diffusion behaviors. But in the case of immobilized enzymes, investigation of the substrate diffusion behavior near the surface is difficult and so diffusion coefficient ‘De’ of

3.1. Tracer diffusion This method is performed with a consideration of a diffusive tracer layer surrounding the nanocrystalline material. A single nanocrystal is 5

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Fig. 3. Enzyme immobilized on porous and surface modified nanocarrier.

4.2. Surface modification by silanizing agent

spherical carrier is commonly determined. Also easy recovery and reuse of catalysts with high catalytic activity by covalent tethering to a support is a long term objective in catalysis research [87]. Fig. 3 depicts the importance of surface porosity in enzyme immobilization. It is clear that in porous carriers, the enzyme diffuses through the pores and occupy various sites, by the same way the substrate has to solve both internal and external diffusion limitations to reach the enzyme. Whereas, it is sufficient to solve only external limitations in the case of surface modified nano particles.

Certain hybrid organic/inorganic catalysts have used along with various porous support materials such as zeolites due to their high surface areas and well- defined structures [92–95]. Additionally, the organic polymers are also extensively used as catalyst supports [96,97] and the recoverable soluble catalysts have gained much interest in recent years because of high diffusion limitations faced by these porous supports. Non-magnetic nanoparticle supported catalysts faces the difficulty in quick and easy recovery after reaction. Seenuvasan et al [98] and Li et al. [99], reported an alternative support using inert barrier by the silica coating for hybrid organic/inorganic sulfonic acid/magnetic nanoparticle catalysts.

4.1. Surface modification in porous nanocarriers In general, the porous nanocarriers are the suitable ones for enzyme immobilization because of high enzyme loading but it is associated with high enzyme loss. At the same time, if the porosity reduced nano carriers are considered for enzyme immobilization, good net result of enzyme activity with negligible enzyme loss can be seen. This is due to the fact of direct relationship between the porosity and the adherence of the enzyme. In this context, surface modification of porous carriers enable minimum enzyme attachment and allow all the available active sites to take part in the catalytic reaction positively. Table 4 details few studies which reported the variation of diffusion limitations through surface modification.

4.3. Surface modification using nanospheres Recent studies proposed a nanosphere for elimination of diffusion limitations inside bulk mesoporous materials. Distinct mass transfer inhibition was expressed by the bulk carbon-supported gold nanocatalysts in comparison with an ordered mesoporous carbon nanospheresupported gold catalysts [100].

Table 4 Variation of diffusion limitations by surface modification of nanoparticles. S.No

Nanoparticles

Surface Modification

Results of Diffusion Limitations Variation

References

1 2 3 4 5 6 7 8

Iron oxide Gold Cadmium selenide Cobalt Silica Graphene nanosheets Iron oxide Dendrimer

Amino coating PEG PEG Carbon coating TEOS/ Organosilane reagents Gold nanoparticles Surface Coating Cross-linking

Enzyme immobilization Photo controlled drug delivery Ligand exchange and dispersion stability Magnetic separation Reduced aggregation and non-specific binding Functionalities and reactive sites Immobilization of laccase for textile recalcitrant degradation Drug (Protein) Immobilization

[73] [87] [88] [89] [90] [91] [12] [56]

6

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4.4. Surface modification using nanosheets

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Zhiani et al., [101] prepared a new, highly accessible and low diffusion limitations nanocatalyst using phosphosilicate nanosheet. Palladium nanoparticles that were supported on fibrous phosphosilicate was employed as nanocatalyst. It was also reported that the decrease in diffusion limitations improves the stability of the nanocarriers. 4.5. Surface modification by amino silanes The surface porosity of magnetite nanoparticles was reported to be altered by amino tagging [73]. Decrease in the diffusion coefficient due to modification in surface porosity resulted in higher enzyme activity. This type of surface modification can be chosen for studies involving the attachments of biomolecules on the nanoparticles [102]. 5. Conclusions Enzymes are widely used in industrial biochemical reactions for their catalytic properties, but they lose their stability on repeated use. Hence stability of the enzymes is increased and are highly utilized by enhancing the activity using immobilization. Diffusion of substrate and the product is very important in the successful catalytic actions of the immobilized enzyme. Among various carriers used in immobilization, nanomaterials serve as an excellent carrier for immobilizing enzyme except for the reason interruption caused by diffusion limitations in porous nanomaterials. Several strategies are implemented to overcome the diffusion limitations out of which surface modification seems to be the best method. This is because of the direct relation between the surface porosity and the diffusion limitations. This review on the diffusion limitations in the nanosystem is believed to support the future catalytic studies with effective elimination of diffusion limitations. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. References [1] W. Addah, J. Baah, T.A. McAllister, Effects of an exogenous enzyme-containing inoculant on fermentation characteristics of barley silage and on growth performance of feedlot steers, Can. J. Anim. Sci. 96 (2016) 1–10. [2] V. Ravindran, Feed enzymes: the science, practice, and metabolic realities, J. Appl. Poult. Res. 22 (2013) 628–636. [3] W.D. Crabb, C. Mitchinson, Enzymes involved in the processing of starch to sugars, Trends Biotechnol. 15 (1997) 349–352. [4] R.L. Harrison, B.C. Bonning, Proteases as insecticidal agents, Toxins. 2 (2010) 935–953. [5] A.A. Bekhit, D.L. Hopkins, G. Geesink, A.A. Bekhit, P. Franks, Exogenous proteases for meat tenderization, Crit. Rev. Food Sci. Nutr. 54 (2014) 1012–1031. [6] A. Georgala, E. Anifantakis, Evolution of lipolysis during the ripening of traditional feta cheese, Food Chem. 93 (2005) 73–80. [7] L. Zhang, Z. Cui, Improvement of the quality and shelf life of wheat bread by a maltohexaose producing α-amylase, J. Cereal Sci. 85 (2019) 305–309. [8] A.J. Das, T. Miyaji, S.C. Deka, Amylolytic fungi in starter cakes for rice beer production, J. Gen. Appl. Microbiol. 63 (2019) 236–245. [9] A.O. Adiguzel, M. Tuncer, Production, characterization and application of a xylanase from streptomyces sp. AOA40 in fruit juice and bakery industries, Food Biotechnol. 30 (2016) 189–218. [10] J.S. Tolan, Pulp and paper, in: T. Godfrey, S. West (Eds.), Industrial Enzymology, 2nd, Stockton Press, New York, 1996, pp. 327–338. [11] V.V.Leo Lallawmsanga, A.K. Passari, I.K. Muniraj, A. Uthandi, A. Hashem, E.F. Abd Allah, A.A. Alqarawi, B.P. Singh, Elevated levels of laccase synthesis by Pleurotus pulmonarius BPSMIO and its potential as a dye decolorizing agent, Saudi J. Biol. Sci. 26 (2019) 464–468. [12] N. Balaji, K.S. Kumar, S. Muthulingam, G. Vinidhini, M.A. Kumar, Immobilization of laccase onto micro-emulsified magnetic nanoparticles for enhanced degradation of a textile recalcitrant, J. Environ. Biol. 37 (2016) 1489–1496.

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