Phytases and their pharmaceutical applications: Mini-review

Journal Pre-proof Phytases and their pharmaceutical applications: Mini-review Archita Sharma, Ojasvini Ahluwalia, Abhishek Dutt Tripathi, Gursharan Singh, Shailendra Kumar Arya PII:

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https://doi.org/10.1016/j.bcab.2019.101439

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BCAB 101439

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Biocatalysis and Agricultural Biotechnology

Received Date: 12 July 2019 Revised Date:

11 November 2019

Accepted Date: 18 November 2019

Please cite this article as: Sharma, A., Ahluwalia, O., Tripathi, A.D., Singh, G., Arya, S.K., Phytases and their pharmaceutical applications: Mini-review, Biocatalysis and Agricultural Biotechnology (2019), doi: https://doi.org/10.1016/j.bcab.2019.101439. 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 Ltd.

Phytases and their pharmaceutical applications: Mini-review Archita Sharma1, Ojasvini Ahluwalia1, Abhishek Dutt Tripathi2, Gursharan Singh3*, Shailendra Kumar Arya1* 1

Department of Biotechnology, University Institute of Engineering and Technology, Panjab

University, Chandigarh, INDIA 2

Centre of Food Science and Technology, Banaras Hindu University, Varanasi, India

3

Department of Medical Laboratory Sciences, Lovely Professional University, Phagwara144411, Punjab, India *Corresponding authors [email protected] [email protected]

Abstract Phytate (myo-inositol hexakisphosphate) is considered as the main source of phosphorous in plants, although it also occurs predominantly in legumes, oilseeds and cereal grains. The nonhydrolyzed phytate when it comes to the excreta of animals as an undigested part of their feed enhanced the level of phosphorous in natural environments and contributes to the accumulation of phosphorus and finally causes the pollution like eutrophication, and greenhouse gases emission. Phytases (myo-inositol hexakisphosphate hydrolases) are very important biocatalysts and significantly use in the animal feed industry to convert phytate to inorganic phosphorous. Many review articles on phytases are available in the literature but very less information is present on their pharmaceutical importance. The present article is describing the role of phytases in controlling diabetes mellitus, atherosclerosis, coronary heart disease and ceasing of stone formation in kidneys, inhibition of human immunodeficiency virus (HIV) and heavy metal toxicities, human and animal nutrition as well. Keywords: Animal nutrition, Anti-cancer agent, Coronary heart disease, Eutrophication, Phytase.

1. Introduction Myo-inositol hexaphosphate phosphohydrolase, i.e. phytase enzyme and belongs to the class of enzyme classes E.C. 3.1.3.8 and E.C. 3.1.2.26, (Neilson et al., 2013) mainly catalyzes the phytate to myo-inositol and inorganic phosphate (Fig. 1, b) and is observed as an important metabolic process in numerous biosystems (Rebello, et al., 2017). The occurrence of phytate is predominantly in the legumes, cereal grains, oilseeds, etc. The increased environmental pollution, particularly the accumulation of phosphorus in the agricultural lands and rivers have increased the awareness in society and thus demanding fresh regulation all over the world to curb this problem. Basically the phytase is considered as the industrial enzyme and also called the boom in feed producing industries with an ability to release the inorganic phosphorous supplements in feed by hydrolyzing the phytate (Rebello, et al., 2017). It has been reported that phytase supplementation can reduce the amount of phosphorus in manure up to 30% approximately. It forms a complex with some proteins that resist the proteolysis (Schlemmer et al., 2009; Liu et al., 2014). Fig 1: Advantages of the phytases if use in food industry (a) reduces the phosphate disposal to the water bodies, (b) preventing the anti-nutritional properties of phytate in body of animals and humans (modified figure from, Rebello, et al. (2017). Most foods of the plant origin contains ~75% of total phosphorus as phytate or phytic acid, whereas other plant parts like roots and tuber have phytate in fewer amounts, ~0.1%. Apart from the phytate, some other inositol phosphates like inositol pentaphosphates, inositol tetraphosphate, etc. are present in seeds but in lower amounts, i.e. <15%. It has been reported that phytase being an ester-hydrolyzing enzyme with an approximate molecular mass in the range of 40–100 kDa is normally active with a pH that ranges from 4.5–6.0 (Vats and Banerjee, 2004). The significant portion of iron which is present in foods like cereals, legumes, etc. is not available for absorption in humans directly because of the formation of complexes with the negatively charged phosphate groups of phytate. Less intake and poor absorption of iron from the diets are the usual reasons for the anemia in young women, pregnant mothers, adults, and the elders which are prone to or potential candidates of anemia (World Health Organisation (WHO), 2008). The phytic acid chelates the iron and thus less uptake of iron or renders them unavailable. The phosphate groups are negatively charged at physiological conditions, which results in the chelation of the cations like iron and zinc. The chelation results in less absorption of iron and zinc (Bohn et al, 2008; Schlemmer et al., 2009). Phytases help in catalyzing the release of the phosphate group from phytate, thus results in the release of the chelated minerals like iron and zinc (Neilson et al., 2013).

In the case of phytase, the maximum research has been done about their involvement in the feed industry especially for the swine and poultry feed. Meanwhile, phytases are very less explored for their potent applications in the pharmaceutical industry. The present review is the first article i.e. providing the information and importance of phytases for their applications in the pharmaceutical industry. 2. Quantity of phytate present in the diet, according to the countries In the case study of the United Kingdom (UK) it has been reported, intake of phytate varies from 504 to 844 mg for adults (Davies et al., 1986), after two decades the intake of phytate was reported 1436±755 mg (Heath et al., 2005). Evaluating 13 Italian diets, an estimate of mean phytate intake was 219 mg/day (Carnovale et., 1987). In India, the average intake of phytate (daily) has a range from 670 to 2500 mg (Grewal et al., 1995). Considering children of the age group 4–9 years the intake was 720-1160 mg. An adolescent with an age group of 10–19 years has a value ranging from 1350-1780 mg (Khokhar et al., 1994). Studies were done by researchers on countries like Sweden and Finland there has been a report of low average intake of phytate 180–370 mg in case of adults when diets with western style are considered (Carnovale et., 1987), whereas for vegetarians the value is comparatively higher for Sweden, that is, 1146 mg (Brune et al., 1989). 3. Sources and some important characteristics of the phytases Phytases are present ubiquitously (Table 1 & 2) in bacteria, fungi, and plants, but for the commercial use of phytases produced from bacteria and fungi only. A Variety of strains of bacteria, yeasts, and fungi have been reported frequently in the literature. Now a day’s animal feed generating industries looking for thermostable or thermotolerant phytases that can fit their process requirements. It has been investigated, the heat-stable phytases can bare the temperature up to 100°C for 20 minutes and still 10% of the enzyme activity was remained (Wang et al., 2007). The presence of phytate in the feed alters the secretion of endogenous compounds such as animal digestive enzymes, HCl (hydrochloric acid), mucin, etc and hence, availability of energy and amino acids are reduced. Phytates are poorly utilized by non-ruminant animals because of the low activity of phytase in their digestive tract. Hence, there is a growing concern over the adverse impact of the anti-nutritional property of phytate on animal performance, phosphorus pollution of effluents from intensive animal operations. As a result, for the last two decades phytate degrading enzyme, phytase from microbial sources has emerged as the primary feed enzyme worldwide. The competence of microbial phytases to release the phytate-bound phosphorous and the potential benefits of this exogenous feed enzyme in improving nutrient digestion and bird performance are well

recognized (Selle and Ravindran, 2007). Due to the less pH of the human stomach and alimentary canal, very low activity of phytase appears in the small intestine, thus approving that the small intestine of human has very restricted potential to digest myo-inositol hexakisphosphate (IP6 ) (Sandberg et al., 2002). 4. Applications of the phytase as an industrial biocatalyst Phytase has a broad spectrum of applications but primarily studied for its uses in food industries to enhance the nutritional values of the product (Fig. 1, b). Phytases are used widely in the United States and Western Europe, where their application is considered to be the industrial standard. The use of phytase preparations as feed supplements has recently increased significantly in China, India, and Southeast Asia. In Russia, the use of phytase preparations is still not particularly popular. However, the development of animal and poultry farming, economic feasibility, and the intensification of programs for ecological protection stimulate this process (Gessler et al., 2018). Phytase is integrated into 90% poultry and 70% swine diets that control phosphorus pollution and improves nutrient uptake. Phytase hydrolyzes the phosphorylated phytic acid that will result in myo-inositol and phosphate (Çinar et al., 2015; Abdulla et al., 2017). Rely upon diet, species, and level of supplementation of phytase; phosphorus excretion can be lowered between a percentage range of 25 and 50% (Haefner et al., 2005). Phytic acid hinders the absorption of iron in foods like cereals and legumes and thus the high prevalence of iron deficiency, for instance, in newborn, fertile women, or vegan community people (Gibson and Hotz, 2001) Phytase reduces the phytic acid content in food products quite effectively. Scientists have investigated that there is the possible use of phytase in bread-making process, also (Rehms and Barz, 1995). 5. The pharmaceutical potential of phytases 5.1. As an antioxidant in food products Oxidation being a damaging mechanism causes hefty loss of nutritive value of the foods. Certain foods with a high content of unsaturated fatty acid and iron are more prone to the oxidation process. With a low percentage of oxygen (<1%), the oxidation reaction still can proceed and may result in unwanted changes in flavor, color, loss of nutritional value and spoilage of the foods. Adding antioxidants will minimize the consequences of the oxidation reaction. With regard to this, phytase is one of the naturally existing antioxidants (Silva and Bracarense, 2016). Out of 22 immediately available iron chelators, the only phytase exist as most potent and non toxic antioxidant in food products (Šnyrychová et al., 2006).

5.2. Phytase as an anti-cancer agent Phytases have several pharmaceutical properties (Fig.2) and an anti-neoplastic agent is one of them (Table 3). The human colon cancer cell line HT-29, human leukemic hematopoietic cell lines, such as K-562 and human normal and leukemic hematopoietic cells were inhibited by the application of phytase. Furthermore, it has the potential to restrict the proliferation of cells like breast cancer, cervical cancer, and prostate cancer in humans (Shamsuddin et al., 2002). The growth of tumors like mesenchymal tumors, murine fibrosarcoma (Raina et al., 2008) and human rhabdomyosarcoma (Tantivejkul et al., 2003) was reduced by consuming foods that are phytase-rich (Vucenik and Shamsuddin, 2003). Fig. 2: Applications of phytases in medical domain treating coronary heart disease, colon cancer, HIV inhibitor 5.2.1. Colon cancer It is one of the main causes of misery and mortality in western countries due to the low intakes of dietary fibers (Nawrocka et al., 2012). Amid the various units of fiber, phytase has been studied immensely for inhibitory results against colon carcinogenesis (Fox et al., 2002). In vitro investigations by researchers have revealed that human colon cancer cells namely human colon adenocarcinoma cell line HT-29, were hindered when treated with phytase (Vucenik and Shamsuddin, 2006). 5.2.2. Breast cancer Plentiful debates have been established that phytase has an inhibitory effect on the mammary carcinoma. But still, there are no conclusive shreds of evidences available because of this the area is almost untouched. It has been suggested that phytase and inositol displayed synergistic effects against the mammary carcinogenesis that results in a 48% decline in the bunch of tumor, also slight decreases in tumor size were noticed when correlated with animals (Shamsuddin and Bose, 2012). 5.2.3. Rhabdomyosarcoma (RMS) It is a tumor of mesenchymal origin. It is the most common sarcoma of soft tissues in children (Vucenik et al., 1998b). Patients with advanced metastatic rhabdomyosarcoma frequently generally do not respond to therapies available at present. In vitro and in vivo investigations have stated that the effects of phytase on the human rhabdomyosarcoma cell line have suppressed the growth in a time-dependent manner and dose-dependent manner Apart from all; it also helps in the induction of differentiation of cells. Additionally, after the removal of phytase from the media, there has been an observation of logarithmic growth

again (Vucenik, Zhang, & Shamsuddin, 1998c). Eventually, phytase has a significant role to play in treating fibrosarcomas in humans due to observation made from past reported data like intraperitoneal injections of phytase in mice which reduces the growth of subcutaneous transplanted murine fibrosarcomas. This results in increasing the survival of mice with a tumor, and also decreases the number of pulmonary metastases (Vucenik et al., 1992). These reported investigations have found out that phytase is a potential candidate for providing therapy in humans against rhabdomyosarcoma and various other mesenchymal neoplasms. 5.2.4 Pancreatic cancer This type of cancer is considered as one such malignancy which is extremely resistant to chemotherapies. The estimated mortalities from pancreatic cancer were 31,270 in the year 2004 (Somasundar et al., 2005). The characteristic of pancreatic adenocarcinoma is its poor prognosis (Vucenik & Shamsuddin, 2003). The insensitivity towards any traditional therapies for treating pancreatic cancer is the resistance to apoptosis. From the already reported data, the in vitro administration of corn-derived phytate and rice-derived phytate on human pancreatic adenocarcinoma cells PANC 1 and MIAPACA has diminished its growth within the range of 37.1–91.5% (Somasundar et al., 2005). Such data have suggested that phytase has the strength to efficiently treating pancreatic cancer. Additionally, in vivo studies and human investigations are required for evaluation of safety and the clinical utility of such agents in patients having pancreatic cancers. 5.3. Against coronary heart disease Coronary heart disease is the leading source of morbidity and mortality from industrialization point of view. It has been found out that approximately 30% of all deaths in the United States are because of this disease (Kumar et al., 2010). Analysis shows that there is a link with elevated plasma cholesterol, leads to hypercholesterolemia. Fiber-rich diets particularly phytase-rich, have the shielding effect of decreasing the ratio of zinc to copper which is absorbed from the intestinal tract because phytase preferentially binds with zinc rather than copper and thereby it can be concluded that phytase will lead to a decrease in the absorption of zinc without having effects on copper absorption (Li et al., 2017). 5.4. Antiplatelet activity of phytases Platelet adhesion to endothelial cells and their aggregates are particular steps in the pathogenesis of thrombosis and atherosclerosis. An investigation of the effect of phytase on platelet aggregation was conducted, with whole blood attained from healthy volunteers and the result was that phytase competently inhibits human platelet aggregation in vitro, that

suggests that it has potential in lowering down the cardiovascular disease (CVD) risks (Nawrocka et al., 2012). 5.5. Phytases against diabetes mellitus Diabetes mellitus is caused by continuous intake of a diet with available glucose and has more prevalence in western culture. This leads to inference in an abnormally high level of blood sugar (hyperglycemia). Withal, foods that are phytase-rich are of great consideration as there is a negative connection that exists among the intake of phytase and blood glucose response. Phytase can be considered as a key element in regulating insulin secretion; decrease the production of insulin etc. The actual mechanism of action is still unclear but phytase helps in regulating insulin secretion along with its effect on calcium channel activity and, in turn, opens calcium channels of intracellular origin, and results in the release of insulin (Vucenik et al., 2004). 5.6. Phytases against HIV Phytase was explored for its antiviral fallout on the human immunodeficiency virus in vitro. It has been seen that phytase completely inhibited, like in MT-4 cells, the cytopathic effect of the human immunodeficiency virus (Bhowmik et al., 2017). Furthermore, phytase also inhibits the replication of HIV-1 in a T cell line, along with that of a freshly isolated strain from peripheral blood single nuclear cells (Kang et al., 2010). Although the mechanisms of inositol phosphate-6 (IP6) action is still unclear and can be figured out that it acts on HIV-1 during an early replicative stage. It is not viable to evolve phytase on its own as an anti-AIDS drug. Reports for this anti-HIV agent might be conventional to show a basis for the consequent production of preferable drugs for the treatment of acquired immunodeficiency syndrome (AIDS). 5.7. Phytases against dental caries Dental caries or tooth decay is considered the most accepted disease over the world. Epidemiological research has shown that there is a significantly increased prevalence of dental caries accompanying the shift in dietary habits in western culture. This boost has led to a conjecture of decreased phytase desolation justified by the high levels of carcinogenicity of flour upon refinement and has attributed to its capacity to lower down calcium, fluoride and phosphate solubilities, particularly enamel constituents (Greiner et al., 2007). Likewise, phytase has a high inclination for hydroxyl-apatite. It treats the formation of cavities, a plaque by just protecting teeth from demineralization. 6. Human health and medicine

Phytase regarding human health and medicine imitates an interesting new outlet (Pagano et al., 2007). There is a non-phosphorus-related benefit of high dietary phytase supplementation in the case of bone development in young pigs. As pigs are the perfect model for humans, it would be exciting to test in case pig results can be transferred to humans or not. It is also being used in solo or in consolidation with other reagents, like strontium, in order to cure or to prevent osteoporosis. A study has suggested that there is a potential act for zinc and phytase in improving the efficacy of botulinum toxin in the analysis of cosmetic facial rhytids, benign essential blepharospasm, and hemifacial spasm (Koshy et al., 2012). 7. Other applications: 7.1. Synthesis of lower inositol phosphates Lower phosphoric esters of myo-inositol have a significant and important part in the signalling of transmembrane processes and in the mobilization of calcium from an intracellular part in the animal also in plant tissues. The use of phytase which is isolated from Aspergillus niger hydrolyzes inositol hexaphosphate 6 (IP6) to lower phosphorylated derivates from pentaphosphate 5 (IP5) to inositol phosphate-2 being dependent on the quantity of enzyme (Vohra et al., 2003). 7.2. Low-phytase crops as an alternative technology Appreciable achievement is now been accomplished in the development of low-phytase crops, for example, maize, barley, rice, and wheat, with the enrolment of mutants of low phytic acid, respectively. With well reported documentation on the efficacy of these lowphytase crops that remodels animal and human nutrition of phosphorus there is a comprehensive approval of low–phytase cultivars that will provide another means to mitigate dietary and environmental dispute emerging from extensive phytase (Lei et al., 2013). Phytase involvement in the regulation of metabolism and physiology of the plant has apparently make phytase important for seed germination, growth, and resistance to disease without absolutely complete removal of the massive amount of phytase in plants (Mazariegos et al., 2006). 7.3 Phytase in food Concerning human consumption, phytase has shown to be a promising candidate in the processing and manufacturing of food. Extensive research has been going on in this very domain for improving the nutritional value of foods obtained from the plant along with the improvement in the processing activity of food, technically. Phytate rich diet results in less absorption of dietary minerals (Lopez et al. 2002) along with phytate dephosphorylation during the processing of food and thus the formation of partially phosphorylated myo-inositol

phosphate esters only having less potential to hinder the intestinal uptake of dietary minerals (Sandberg et al. 1999). Phytase has the strength to produce bread with low phytin (Simell et al. 1989). Additionally, phytase enhances the quality of bread in two ways; firstly, by improving the nutritional value by diminishing the content of phytate, secondly, by activating endogenous α-amylase with more availability of calcium (Haros et al., 2001). With the enhancement in the bioavailability of minerals and trace elements, adding phytase during the food processing activity has also been documented in bread making (Haros et al. 2001), production of plant protein isolates (Fredrikson et al. 2001), corn wet milling (Caransa et al. 1998) and the fractionation of cereal bran (Kvist et al. 2005) which affects the economy of the production process, yield, and quality of the final products in a positive way (Griener and Konietzny, 2006). Apart from decreasing the content of phytase in doughs, fresh bread, etc., the duration of fermentation cut down by adding phytase with no or negligible effect on the pH of dough. Also, the volume of bread with the improved texture of crumbs has also been noticed. All the formulations have reduced firmness, hardness, gumminess, and chewiness thus soft crumbs when the dough was supplemented with phytase. Such improvements have suggested the indirect effect of phytase on the activity of α-amylase activity. Despite all the advancements and improvements, the total removal of phytate has not been accomplished. One of the examples of phytase in human nutrition is given below (Griener and Konietzny, 2006). 7.3.1 Doli Ki Roti: An example of indigenous fermented Indian bread Chapathi (generally call as roti) is the staple food in the northern states of India and some neighboring countries. It is prepared from whole wheat flour that contains enough amounts of phytic acid. The reduction of the phytate level within chapattis is possible through fermentation of the wheat flour. A mutated strain of the yeast Candida versatilis, as a source of phytase, was recommended during the preparation of chapatti dough, which decreases the level of phytate by 10–45% (Bindu & Varadaraj, 2005). In India, numerous fermented foods such as idli, dosa, dhokla, vada, etc. are manufactured and consumed by a large group of population. Doli ki roti is also an example of indigenous fermented Indian bread based on the wheat and is prepared by the Indian Punjabi community migrated from Western Pakistan during partition. During the preparation of Roti, an earthen pot is being used when the fermentation process is being carried out and known as doli, and thus the name Doli ki roti (fig.3). Fig. 3: Preparation method of Doli ki roti (Bhatia et al. 2012)

This particular indigenous fermented and stuffed bread is an ideal amalgamation of both cereals and legumes and apart from this; it is very nutritive because of the cumulative impact of germination and fermentation. It has less amount of phytic acid and high availability of less dietary minerals, with an impact of fermentation that reduces the phytate level in Roti (Bhatia et al. 2012). 7.4. Vanadate substituted phytase Phytase with a substitution of vanadate has grabbed the attention of numerous researchers due to their capability to act as peroxidase. This substituted phytase behaves like an oxygen transfer catalyst in sulfide oxidations (van de et al. 2000). Taking into consideration all the enzymes, vanadium haloperoxidases has the potential to utilize them in the applications of catalytic oxidative transformations. The enzyme vanadium haloperoxidases are structurally similar to the histidine acid phosphatises, that is phytases with a strength to carry forward the phosphatase reactions by replacing vanadate with phosphate (Renirie et al. 2000). Vanadium is considered as a phosphatase inhibitor and due to structural similarities vanadate acts as an analog of the transition state and results in the formation of a trigonal bi-pyramidal complex with the presence of nucleophilic histidine at the active site of acid phosphatises (Davies and Hol, 2004). In the year 2002, it has been reported that when acid phosphatase was incubated with vanadate it acts as a peroxidase (Tanaka et al. 2002). In spite of the fact that not much research effort has been done in this direction regarding the exploitation of enzyme hemeperoxidases in industrial oxidative processes, application of heme-peroxidases is inhibited due to inactivation via the presence of excess hydrogen peroxide (H2O2) and limited substrate amount (van Deurzen et al. 1997). A group of researchers have performed an experiment and investigated the ability of vanadium-phytase-cross linked enzyme aggregates (CLEA) to function as a heterogeneous and recyclable catalyst in the sulfoxidation reaction of a model substrate named methylphenyl sulfide. This vanadium-phytase-cross linked enzyme aggregates (CLEA) catalyst has high conversion value, rational selectivity, better recyclability under aqueous conditions, etc. Nonetheless, when more than 10 percent of organic solvent is present, there has been a decrease in the activity of the catalyst. Hence, investigations have been done to exemplify the role of vanadate on the active site and on the effect of organic solvents in the conformation of the enzyme. 51V NMR was exploited to investigate the plausible intermediates of vanadiumperoxidase for both 3-phytase and 6-phytase, respectively. Circular dichroism was utilized to determine the conformation of enzymes in various solvents and at different temperatures. The 6-phytase was inactive in the sulfoxidation of thioanisole has a high affinity for vanadate at

pH 7.6 as compared to the pH at 5.0 and the situation for 3-phytase was just the opposite of 6-phytase. Inclusively, the data of nuclear magnetic resonance (NMR) of vanadium substantiate the hypothesis that vanadium is covalently linked with the active site of the enzyme to an apical histidine and to oxygen donors. When hydrogen peroxide is added, there is a formation of two peroxide-vanadate-phytase complexes at pH 5.0 in the case of 3-phytase whereas in case of 6-phytase, there is no such formation of peroxide-vanadate-phytase complexes due to the inactivity of the 6-phytase for the oxidation of thioanisole (Correia et al. 2008). Another group investigated the oxidation of thioanisole with hydrogen peroxide catalyzed by phytase from Aspergillus ficuum with the incorporation of vanadium in it. With low concentrations of vanadate (<15 µM), there has been a quantitative conversion into sulfoxide (with a preference of S-enantiomer at 56% ee) whereas at high concentration of vanadate (> 25 µM), there has been an additional oxidation of respective sulfone (van de Velde et al. 1998). 8. Conclusions Despite the ample economic interest, low yield and high synthesis cost of phytases are one of the limiting factors before using this enzyme in the animal diet and pharma industry. There is a need for more research and development to find out the cost-effective methods for the production of microbial phytases. The constructive health effects of phytase are important for populations of developed countries as a result of the higher cases of cancer particularly colon cancer which is connected with higher fat and intake of lower fiber-rich food. Such a wide topic implies that more in-depth studies are required with an understanding of the mechanism. 9. Future perspectives An extensive swing in phytase research is the screening of enzymes of thermal stability with higher value. Till now, only a few phytases have been documented with the stability of temperature or optimal temperature exceeding 70°C. The focus is to remodel the industrial production of phytase related to the cost-effective level and will persist with this as the efforts of using phytase in feed and food industries have been much more successful. Conflict of interest: There is no conflict of interest.

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Table 1: Intake of dietary phytate (daily) in numerous countries (Schlemmer et al. 2009) Country

Conditions

Daily phytate intake (mg) (mean value, mean±SD or range)

UK

Male (>40 years) Male–female Male–female Male–female (North-West) Male–female (North-East) Male–female Male–female (35–76 years) Western type diets Male–female (35–76 years) vegetarian diets Male–female

1436±755 600±800 219 (112–1367) 288 320 180 369 (230–532)

Europe

Italy

Sweden

Finland North and Central America USA

Canada Asia India Thailand

1146 (500–2927) 370

Infants (<1 years) Children (4–5 years) Male–female (19–35 years) Female (4–5 years) Male (4–5 years)

166 ± 167 501±271 1293 ±666 250 (132–318) 320 (203–463)

Male–female Male–female (>45 years) Females nonurban Males nonurban

670 1290–2080 1139 ± 481 1104 ± 965

Table 2: Different microbial sources of phytases with production method (Pandey et al., 2001) (SmF= Submerged Fermentation, SSF= Solid State Fermentation)

S.No

Different sources

Temp. & pH optimal conditions for enzyme activity

References

Prokaryotes 1 2

Bacillus amyloliquefaciens Bacillus subtilis

3

Serratia sp.PSB-15, Enterobacter cloacae strain PSB-45 Escherichia coli (gene expressed in

4

Neutral pH range 55-70oC

(Idriss et al., 2002) (Kerovuo et al., 2000) (Lei et al., 2007)

50–70/ 3 to 8

(Kalsi et al., 2016)

50/ 4.0-60

(Tai et al., 2013)

50/04 60/6.5

(Mopera et al., 2012) (Kerovuo et al., 2000)

Pichia pastoris)

5 7

Klebsiella pneumoniae 9-3B Bacillus subtilis

Eukaryotes 13

Fungi Aspergillus tubingensis

NA

(Qasim et al., 2016)

14 15 16

Aspergillus fumigatus gene expressed in Pichia pastoris A. niger PhyA

20

A. niger SK-57 Sporotrichum thermophile

21

Plants Histidine acid phosphatases

22

Lilly pollen phytase

23

Animals Brush border vesicles of poultry

24 25

Hybrid striped bass Yeast Pichia anomala

37/2.5

(Rodriguez et al., 2000)

2.0 and 5.0-5.5 55-60oC 37 to53/2.5-5.5

(Ahmad et al., 2000) (Lei et al., 2007) (Casey &Walsh, 2003)

60oC, pH=5.0, 456 kDa

Singh & Satyanarayana (2010)

4.5-6.0 38-55oC 8.0 55 oC

(Lei et al., 2007)

5.5-6.0

(Lei et al., 2007)

3.5-4.5

(Lei et al., 2007)

64kDa, 60 oC, pH=4.0

(Vohra & Satyanarayana 2002)

(Lei et al., 2007)

Table 2. Example of transgenic plants with the expression of phytases (Singh and Satyanarayana, 2011) Source of phytase Aspergilus niger

• • • •

Expression in Wheat Arabidopsis Canola Maize

• • • •

References Brinch-Pedersen et al. 2000 Mudge et al. 2003 Ponstein et al. 2002 Chen et al. 2008

Aspergilus fumigatus Escherichia coli

Tobacco Rice

Wang et al. 2007 Hong et al. 2004

Selenomonas ruminantium

Rice

Hong et al. 2004

Bacillus subtilis

Tobacco

Yip et al. 2003

Bacillus subtilis

Lung et al. 2005

Aspergillus sp.

Tobacco Arabidopsis Maize

Drakakaki et al. 2005

Medicago truncatula

Arabidopsis

Xiao et al. 2005

Table 3: Phytase as an anti-cancer agent (Kumar et al. 2010) Impact of phytase Phytase against colon cancer

Mode of action Production of butyric acid via fermentation & thus decrease in pH in gut region & metabolism of bile acids.

References Midorikawa et al. (2001) Shamsuddin (2002)

Phytase against mammary carcinoma

Phytase for hepatocellular carcinoma

Phytase for prostate cancer cells (PC3 cells & DU145 prostate cancer cells) Phytase for rhabdomyosarcoma (RMS) Phytase for pancreatic cancer

Phytase for blood and bone marrow cancer

Inhibition of oxidative reactions mediated by iron. Up-regulation of the expression of tumour suppressor genes such as p53, p21 WAF1/Cip1. Stimulation of apoptosis of human breast Vucenik & Shamsuddin (2003) cancer cells. Synergistic role of phytate and inositol on cell division arrest. Improves the activity of tumour suppressor Vucenik et al. (1998a) genes. Differentiation of malignant cells & transformation of cancer cells to less aggressive phenotypes Impairment of both receptor-mediated (EGFR and erbB1) & fluid-phase Zi et al. (2000) endocytosis. Inhibition of mitogenic signals. Suppression of cancerous cells. Vucenik et al. (1998c) Induction of cell differentiation Somasundar et al. (2005) Adjunct of treating pancreatic cancer. Enhancement in the sensitivity with respect to traditional therapies Enhanced differentiation of carcinoma Shamsuddin et al. (1992) cells. Improved and better synthesis of haemoglobin.