Abstract
Background: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus.
Objective: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells.
Methods: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents.
Results: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate.
Conclusion: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed.
Keywords: Phytase, Yersinia intermedia, emzyme kinetics, recombinant enzyme, protein structure, fluorescence quenching.
Graphical Abstract
[1]
Valeeva LR, Nyamsuren C, Sharipova MR, Shakirov EV. Heterologous expression of secreted bacterial BPP and HAP phytases in plants stimulates Arabidopsis thaliana growth on phytate. Front Plant Sci 2018; 9: 186.
[2]
Gaxiola RA, Edwards M, Elser JJ. A transgenic approach to enhance phosphorus use efficiency in crops as part of a comprehensive strategy for sustainable agriculture. Chemosphere 2011; 84(6): 840-5.
[3]
Gontia I, Tantwai K, Rajput LPS, Tiwari S. Transgenic plants expressing phytase gene of microbial origin and their prospective application as feed. Food Technol Biotechnol 2012; 50(1): 3.
[5]
Vashishth A, Ram S, Beniwal V. Cereal phytases and their importance
in improvement of micronutrients bioavailability. 3 Biotech 2017; 7(1): 42.
[6]
Turner BL, Paphazy MJ, Haygarth PM, McKelvie ID. Inositol phosphates in the environment. Philos Trans R Soc Lond B Biol Sci 2002; 357(1420): 449-69.
[7]
Pakbaten B, Majidzadeh Heravi R, Kermanshahi H, Sekhavati MH, Javadmanesh A, Mohammadi Ziarat M. Production of Phytase Enzyme by a Bioengineered Probiotic for Degrading of Phytate Phosphorus in the Digestive Tract of Poultry. Probiotics Antimicrob Proteins 2019; 11(2): 580-7.
[8]
Holm PB, Kristiansen KN, Pedersen HB. Transgenic approaches in commonly consumed cereals to improve iron and zinc content and bioavailability. J Nutr 2002; 132(3): 514S-6S.
[9]
Morgan NK, Walk CL, Bedford MR, Burton EJ. Contribution of intestinal- and cereal-derived phytase activity on phytate degradation in young broilers. Poult Sci 2015; 94(7): 1577-83.
[10]
Shah PC, Kumar VR, Dastager SG, Khire JM. Phytase production by Aspergillus niger NCIM 563 for a novel application to degrade organophosphorus pesticides. AMB Express 2017; 7(1): 66.
[11]
Scholey D, Burton E, Morgan N, Sanni C, Madsen CK, Dionisio G, et al. P and Ca digestibility is increased in broiler diets supplemented with the high-phytase HIGHPHY wheat. Animal 2017; 11(9): 1-7.
[12]
Jain J, Singh B. Phytase Production and development of an ideal dephytinization process for amelioration of food nutrition using microbial phytases. Appl Biochem Biotechnol 2017; 181(4): 1485-95.
[13]
Kumar V, Yadav AN, Verma P, Sangwan P, Saxena A, Kumar K, et al. beta-Propeller phytases: Diversity, catalytic attributes, current developments and potential biotechnological applications. Int J Biol Macromol 2017; 98: 595-609.
[14]
Watanabe T, Ikeda H, Masaki K, Fujii T, Iefuji H. Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris. J Biosci Bioeng 2009; 108(3): 225-30.
[15]
Reddy CS, Vani K, Pandey S, Vijaylakshmi M, Kaul T. Manipulating microbial phytases for heterologous expression in crops for sustainable nutrition. Ann Plant Sci 2013; 2(10): 436-54.
[16]
Nielsen PH, Wenzel H. Environmental assessment of Ronozyme® P5000 CT phytase as an alternative to inorganic phosphate supplementation to pig feed used in intensive pig production. Int J Life Cycle Assess 2007; 12(7): 514.
[17]
Han N, Miao H, Yu T, Xu B, Yang Y, Wu Q, et al. Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation. BMC Biotechnol 2018; 18(1): 36.
[18]
Sanangelantoni AM, Malatrasi M, Trivelloni E, Visioli G, Agrimonti C. A novel beta-propeller phytase from the dioxin-degrading bacterium Sphingomonas wittichii RW-1. Appl Microbiol Biotechnol 2018; 102(19): 8351-8.
[19]
Carrillo Rincon AF, Magdevska V, Kranjc L, Fujs S, Muller R, Petkovic H. Production of extracellular heterologous proteins in Streptomyces rimosus, producer of the antibiotic oxytetracycline. Appl Microbiol Biotechnol 2018; 102(6): 2607-20.
[20]
Huang H, Luo H, Yang P, Meng K, Wang Y, Yuan T, et al. A novel phytase with preferable characteristics from Yersinia intermedia. Biochem Biophys Res Commun 2006; 350(4): 884-9.
[21]
Lei XG, Stahl CH. Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 2001; 57(4): 474-81.
[22]
Holman WI. A new technique for the determination of phosphorus by the molybdenum blue method. Biochem J 1943; 37(2): 256-9.
[23]
Shi XW, Sun ML, Zhou B, Wang XY. Identification, characterization, and overexpression of a phytase with potential industrial interest. Can J Microbiol 2009; 55(5): 599-604.
[24]
Li Z, Zhao A, Wang X, Jin X, Li J, Yu M. Cloning, overexpression, and functional characterization of a phytase from the genus bacillus. J Mol Microbiol Biotechnol 2013; 23(3): 193-202.
[25]
Huang H, Luo H, Wang Y, Fu D, Shao N, Wang G, et al. A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Appl Microbiol Biotechnol 2008; 80(3): 417-26.
[26]
Ushasree MV, Vidya J, Pandey A. Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression. Biotechnol Lett 2014; 36(1): 85-91.
[27]
Kim T, Mullaney EJ, Porres JM, Roneker KR, Crowe S, Rice S, et al. Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive. Appl Environ Microbiol 2006; 72(6): 4397-403.
[28]
Short JM, Kretz KA. Phytase expression systems and methods
of making and using them, US7232677B2 . 2007.
[29]
Bin Y, Huoqing H, Peilong Y, et al. Alga-derived
phytase and gene and application thereof. CN101914506B . 2012.
[30]
Short J, Kretz K, Gray K, et al. O'Donoghue,
E., Mather, E. Recombinant phytases and methods of making and
using them. US20040091968A1 . 2004.
[31]
Lanahan MB, Koepf E, Kretz K. Microbially-expressed thermotolerant
phytase for animal feed. US7252983B2 . 2007.
[34]
Akbarzadeh A, Dehnavi E, Aghaeepoor M, Amani J. Optimization of recombinant expression of synthetic bacterial phytase in pichia pastoris using response surface methodology. Jundishapur J Microbiol 2015; 8(12): e27553.
[35]
Borgi MA, Khila M, Boudebbouze S, Aghajari N, Szukala F, Pons N, et al. The attractive recombinant phytase from Bacillus licheniformis: Biochemical and molecular characterization. Appl Microbiol Biotechnol 2014; 98(13): 5937-47.
[36]
Miao Y, Xu H, Fei B, Qiao D, Cao Y. Expression of food-grade phytase in Lactococcus lactis from optimized conditions in milk broth. J Biosci Bioeng 2013; 116(1): 34-8.
[37]
Guerrero-Olazaran M, Rodriguez-Blanco L, Carreon-Trevino JG, Gallegos-Lopez JA, Viader-Salvado JM. Expression of a Bacillus phytase C gene in Pichia pastoris and properties of the recombinant enzyme. Appl Environ Microbiol 2010; 76(16): 5601-8.
[38]
Zhao Q, Liu H, Zhang Y, Zhang Y. Engineering of protease-resistant phytase from Penicillium sp.: high thermal stability, low optimal temperature and pH. J Biosci Bioeng 2010; 110(6): 638-45.
[39]
Fasimoye FO, Olajuyigbe FM, Sanni MD. Purification and characterization of a thermostable extracellular phytase from Bacillus licheniformis PFBL-03. Prep Biochem Biotechnol 2014; 44(2): 193-205.
[40]
Tai HM, Yin LJ, Chen WC, Jiang ST. Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties. J Agric Food Chem 2013; 61(25): 6007-15.
[41]
Tran TT, Hashim SO, Gaber Y, Mamo G, Mattiasson B, Hatti-Kaul R. Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability. J Inorg Biochem 2011; 105(7): 1000-7.
[42]
Moreira KA, Herculano PN, et al. Optimization of phytase production by Aspergillus japonicus Saito URM 5633 using cassava bast as substrate in solid state fermentation. Afr J Microbiol Res 2014; 8(9): 929-38.
[43]
Casey A, Walsh G. Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142. Bioresour Technol 2003; 86(2): 183-8.
[44]
Wang Y, Gao X, Su Q, Wu W, An L. Cloning, expression, and enzyme characterization of an acid heat-stable phytase from Aspergillus fumigatus WY-2. Curr Microbiol 2007; 55(1): 65-70.
[45]
Santos T, Connolly C, Murphy R. Trace element inhibition of phytase activity. Biol Trace Elem Res 2015; 163(1-2): 255-65.
[46]
Lee SH, Cho J, Bok J, Kang S, Choi Y, Lee PC. Characterization, gene cloning, and sequencing of a fungal phytase, PhyA, from Penicillium oxalicum PJ3. Prep Biochem Biotechnol 2015; 45(4): 336-47.
[47]
Cho JS, Lee CW, Kang SH, Lee JC, Bok JD, Moon YS, et al. Purification
and characterization of a phytase from Pseudomonas syringae
MOK1. Curr Microbiol 2003; 47(4): 290-4.
[48]
National Research Council (U.S.). Subcommittee on Poultry Nutrition.Nutrient requirements of poultry. 9th rev. ed . Washington,
D.C.: National Academy Press 1994; Xiii: p. 155.
[49]
Karimi A, Sadeghi G, Vaziry A. The effect of copper in excess of
the requirement during the starter period on subsequent performance
of broiler chicks. J Appl Poult Res 2011; 20(2): 203-9.
[50]
Pesti GM, Bakalli RI. Studies on the effect of feeding cupric sulfate pentahydrate to laying hens on egg cholesterol content. Poult Sci 1998; 77(10): 1540-5.
[51]
Fugthong A, Boonyapakron K, Sornlek W, Tanapongpipat S, Eurwilaichitr L, Pootanakit K. Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris. Protein Expr Purif 2010; 70(1): 60-7.
[52]
Rodriguez E, Mullaney EJ, Lei XG. Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme. Biochem Biophys Res Commun 2000; 268(2): 373-8.
[53]
Rodriguez E, Wood ZA, Karplus PA, Lei XG. Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. Arch Biochem Biophys 2000; 382(1): 105-12.
[54]
Golovan S, Wang G, Zhang J, Forsberg CW. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can J Microbiol 2000; 46(1): 59-71.
[55]
Laskowski M, Qasim M. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim Biophys Acta 2000; 1477(1): 324-37.
Article Metrics
16
5
2
1