Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges

Hao  Tan; Renyun  Miao; Tianhai  Liu; Xuelian  Cao; Xiang  Wu; Liyuan  Xie; Zhongqian  Huang; Weihong  Peng; Bingcheng  Gan

doi:10.4014/jmb.1604.04051

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Biotechnology and Bioengineering (BB) | Enzyme, Protein Engineering, and Metabolic Engineering

Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges

Hao Tan ^{1, 2}, Renyun Miao ^{1, 2}, Tianhai Liu ^{1, 2}, Xuelian Cao ^{1, 2}, Xiang Wu ^{1, 2}, Liyuan Xie ^{1, 2}, Zhongqian Huang ^{1, 2}, Weihong Peng ^{1, 2} and Bingcheng Gan ^{1, 2*}

¹Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. China, ²Scientific Observing and Experimental Station of Agro-microbial Resource and Utilization in Southwest China, Ministry of Agriculture, Chengdu 610066, P.R. China

Received: April 19, 2016; Accepted: June 29, 2016

J. Microbiol. Biotechnol. 2016; 26(10): 1717-1722

Published October 28, 2016 https://doi.org/10.4014/jmb.1604.04051

Abstract

A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (<44%) to all the known phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at physiological temperature (37°C) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.

Keywords

phytase, acidophilic, acidostable, site-directed mutagenesis, disulfide bridge, thermostable

References

Ariza A, Moroz OV, Blagova EV, Turkenburg JP, Waterman J, Roberts SM, et al. 2013. Degradation of phytate by the 6phytase from Hafnia alvei: a combined structural and solution study. PLoS One 8: e65062.
Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISSMODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.
Bei JL, Chen Z, Fu J, Jiang ZY, Wang JW, Wang XZ. 2009. Structure-based fragment shuffling of two fungal phytases for combination of desirable properties. J. Biotechnol. 139:186-193.
Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42: W252-W258.
Brinch-Pedersen H, Madsen CK, Holme IB, Dionisio G. 2014. Increased understanding of the cereal phytase complement for better mineral bio-availability and resource management. J. Cereal Sci. 59: 373-381.
Cang L. 2004. Heavy metals pollution in poultry and livestock feeds and manures under intensive farming in Jiangsu Province, China. J. Environ. Sci. 16: 371-374.
Chambers JE, Tavender TJ, Oka OB, Warwood S, Knight D, Bulleid NJ. 2010. The reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by Ero1α. J. Biol. Chem. 285: 2920029207.
Craig DB, Dombkowski AA. 2013. Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC Bioinformatics 14: 346.
Dombkowski AA. 2003. Disulfide by Design™: a computational method for the rational design of disulfide bonds in proteins. Bioinformatics 19: 1852-1853.
Dombkows ki A A, S ultana K Z, C raig DB. 2014. P rotein disulfide engineering. FEBS Lett. 588: 206-212.
Emanuelsson O, Brunak S, von Heijne G, Nielsen H. 2007. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2: 953-971.
Fu D, Huang H, Luo H, Wang Y, Yang P, Meng K, et al. 2008. A highly pH-stable phytase from Yersinia kristeensenii:cloning, expression, and characterization. Enzyme Microb. Technol. 42: 499-505.
Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, et al. 2004. Enhancing the thermal tolerance and gas tric p erformance of a m icrobial p hytas e for use as a phosphate-mobilizing monogastric-feed supplement. Appl. Environ. Microbiol. 70: 3041-3046.
Gu W-N, Huang H-Q, Yang P-L, Luo H-Y, Meng K, Wang Y-R, Yao B. 2007. Gene cloning, expression and characterization of a novel phytase from Hafnia alvei. Chin. J. Biotechnol. 23:1017-1021.
Guo C, Diao H, Lian Y, Yu H, Gao H, Zhang Y, Lin D. 2009. Recombinant expression and characterization of an epididymisspecific antimicrobial peptide BIN1b/SPAG11E. J. Biotechnol. 139: 33-37.
Guo C, Liu Y, Yu H, Du K, Gan Y, Huang H. 2016. A novel strategy for thermostability improvement of trypsin based on N-glycosylation within the Ω-loop region. J. Microbiol. Biotechnol. 26: 1163-1172.
Hatala JA, Detto M, Sonnentag O, Deverel SJ, Verfaillie J, Baldocchi DD. 2012. Greenhouse gas (CO2, CH4, H2O) fluxes from drained and flooded agricultural peatlands in the Sacramento-San Joaquin Delta. Agric. Ecosyst. Environ. 150: 1-18.
Hesampour A, Siadat SER, Malboobi MA, Mohandesi N, Arab SS, Ghahremanpour MM. 2015. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis. Appl. Biochem. Biotechnol. 175: 2528-2541.
Huang H, Luo H, Wang Y, Fu D, Shao N, Wang G, et al. 2008. A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Appl. Microbiol. Biotechnol. 80: 417-426.
Inoue H, Fujii T, Yoshimi M, Taylor LE II, Decker SR, Kishishita S, et al. 2013. Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. J. Ind. Microbiol. Biotechnol. 40: 823-830.
Jermutus L, Tessier M, Pasamontes L, van Loon A, Lehmann M. 2001. Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case. J. Biotechnol. 85: 15-24.
Kim M-S, Lei X. 2008. Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR. Appl. Microbiol. Biotechnol. 79: 69-75.
Knox SH, Sturtevant C, Matthes JH, Koteen L, Verfaillie J, Baldocchi D. 2015. Agricultural peatland restoration: effects of land-use change on greenhouse gas (CO2 and CH4) fluxes in the Sacramento-San Joaquin Delta. Global Change Biol. 21:750-765.
Le QAT, Joo JC, Yoo YJ, Kim YH. 2012. Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol. Bioeng. 109: 867-876.
Lei X-G, Weaver JD, Mullaney EJ, Ullah AH, Azain MJ. 2013. Phytase, a new life for an “old” enzyme. Annu. Rev. Anim. Biosci. 1: 283-309.
Liao Y, Li C-M, Chen H, Wu Q, Shan Z, Han X-Y. 2013. Site-directed mutagenesis improves the thermostability and catalytic efficiency of Aspergillus niger N25 phytase mutated by I44E and T252R. Appl. Biochem. Biotechnol. 171: 900-915.
Markowitz VM, Ivanova NN, Szeto E, Palaniappan K, Chu K, Dalevi D, et al. 2008. IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res. 36:D534-D538.
Markowitz VM, Mavromatis K, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. 2009. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 25: 2271-2278.
Merchant HA, McConnell EL, Liu F, Ramaswamy C, Kulkarni R P, B as it A W, M urdan S. 2 011. Ass es sment o f gastrointestinal pH, fluid and lymphoid tissue in the guinea pig, r abbit and pig, a nd implications for t heir us e in drug development. Eur. J. Pharm. Sci. 42: 3-10.
Miller RL, Fujii R. 2009. Plant community, primary productivity, and environmental conditions following wetland reestablishment in the Sacramento-San Joaquin Delta, California. Wetl. Ecol. Manag. 18: 1-16.
Nichols on FA, C hambers BJ, W illiams JR, U nwin R J. 1 999. Heavy metal contents of livestock feeds and animal manures in England and Wales. Bioresour. Technol. 70: 23-31.
Pandee P, Summpunn P, Wiyakrutta S, Isarangkul D, Meevootisom V. 2011. A thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris. J. Microbiol. 49: 257-264.
Patil KR, Roune L, McHardy AC. 2012. The PhyloPythiaS web server for taxonomic assignment of metagenome sequences. PLoS One 7: e38581.
Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Sanchez-Romero I, Ariza A, Wilson KS, Skjøt M, Vind J, De Maria L, et al. 2013. Mechanism of protein kinetic stabilization by engineered disulfide crosslinks. PLoS One 8: e70013.
Shivange AV, Dennig A, Schwaneberg U. 2014. Multi-site saturation by OmniChange yields a pH- and thermally improved phytase. J. Biotechnol. 170: 68-72.
Shivange AV, Serwe A, Dennig A, Roccatano D, Haefner S, Schwaneberg U. 2012. Directed evolution of a highly active Yersinia mollaretii phytase. Appl. Microbiol. Biotechnol. 95: 405-418.
Sone M, Kishigami S, Yoshihisa T, Ito K. 1997. Roles of disulfide bonds in bacterial alkaline phosphatase. J. Biol. Chem. 272: 6174-6178.
Tan H, Wu X, Xie L, Huang Z, Gan B, Peng W. 2015. Cloning, overexpression, and characterization of a metagenomederived phytase with optimal activity at low pH. J. Microbiol. Biotechnol. 25: 930-935.
Tan H, Wu X, Xie L, Huang Z, Peng W, Gan B. 2016. Identification and characterization of a mesophilic phytase highly resilient to high-temperatures from a fungus-garden associated metagenome. Appl. Microbiol. Biotechnol. 100: 22252241.
Tian YS, Peng RH, Xu J, Zhao W, Gao F, Fu XY, et al. 2011. Semi-rational site-directed mutagenesis of phyI1s from Aspergillus niger 113 at two residue to improve its phytase activity. Mol. Biol. Rep. 38: 977-982.
Ullah AHJ, Mullaney EJ. 1996. Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase. Biochem. Biophys. Res. Commun. 227: 311-317.
Vats P, Banerjee UC. 2005. Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem. J. Ind. Microbiol. Biotechnol. 32: 141-147.
Viader-Salvado JM, Gallegos-Lopez JA, Carreon-Trevino JG, Castillo-Galvan M, Rojo-Dominguez A, Guerrero-Olazaran M. 2010. Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris. Appl. Environ. Microbiol. 76: 6423-6430.
Vogt G, Argos P. 1997. Protein thermal stability: hydrogen bonds or internal packing? Fold Des. 2: S40-S46.
Wu T-H, Chen C-C, Cheng Y-S, Ko T-P, Lin C-Y, Lai H-L, et al. 2014. Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design. J. Biotechnol. 175: 1-6.
Yao MZ, Wang X, Wang W, Fu YJ, Liang AH. 2013. Improving the thermostability of Escherichia coli p hytas e, appA, by enhancement of glycosylation. Biotechnol. Lett. 35:1669-1676.
Zhang GQ, Dong XF, Wang ZH, Zhang Q, Wang HX, Tong JM. 2010. Purification, characterization, and cloning of a novel phytase with low pH optimum and strong proteolysis resistance from Aspergillus ficuum NTG-23. Bioresour. Technol. 101: 4125-4131.
Zhang WM, Mullaney EJ, Lei XG. 2007. Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase. Appl. Environ. Microbiol. 73:3069-3076.
Zhao QQ, Liu HL, Zhang Y, Zhang YZ. 2010. Engineering of protease-resistant phytase from Penicillium sp.: high thermal stability, low optimal temperature and pH. J. Biosci. Bioeng. 110: 638-645.
Zhu WH, Qiao DR, Huang M, Yang G, Xu H, Cao Y. 2010. Modifying thermostability of AppA from Escherichia coli. Curr. Microbiol. 61: 267-273.

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Article

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J. Microbiol. Biotechnol. 2016; 26(10): 1717-1722

Published online October 28, 2016 https://doi.org/10.4014/jmb.1604.04051

Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges

Hao Tan ^{1, 2}, Renyun Miao ^{1, 2}, Tianhai Liu ^{1, 2}, Xuelian Cao ^{1, 2}, Xiang Wu ^{1, 2}, Liyuan Xie ^{1, 2}, Zhongqian Huang ^{1, 2}, Weihong Peng ^{1, 2} and Bingcheng Gan ^{1, 2*}

¹Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. China, ²Scientific Observing and Experimental Station of Agro-microbial Resource and Utilization in Southwest China, Ministry of Agriculture, Chengdu 610066, P.R. China

Received: April 19, 2016; Accepted: June 29, 2016

Abstract

A novel phytase of Acidobacteria was identified from a soil metagenome, cloned,
overexpressed, and purified. It has low sequence similarity (<44%) to all the known phytases.
At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at
physiological temperature (37°C) and could retain 92% residual activity after 30 min,
indicating the phytase is acidophilic and acidostable. However the phytase shows poor
stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned
using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time
of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the
wild-type, and its activity and acidostability are not significantly affected.

Keywords: phytase, acidophilic, acidostable, site-directed mutagenesis, disulfide bridge, thermostable

Abstract

References

Ariza A, Moroz OV, Blagova EV, Turkenburg JP, Waterman J, Roberts SM, et al. 2013. Degradation of phytate by the 6phytase from Hafnia alvei: a combined structural and solution study. PLoS One 8: e65062.
Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISSMODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.
Bei JL, Chen Z, Fu J, Jiang ZY, Wang JW, Wang XZ. 2009. Structure-based fragment shuffling of two fungal phytases for combination of desirable properties. J. Biotechnol. 139:186-193.
Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42: W252-W258.
Brinch-Pedersen H, Madsen CK, Holme IB, Dionisio G. 2014. Increased understanding of the cereal phytase complement for better mineral bio-availability and resource management. J. Cereal Sci. 59: 373-381.
Cang L. 2004. Heavy metals pollution in poultry and livestock feeds and manures under intensive farming in Jiangsu Province, China. J. Environ. Sci. 16: 371-374.
Chambers JE, Tavender TJ, Oka OB, Warwood S, Knight D, Bulleid NJ. 2010. The reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by Ero1α. J. Biol. Chem. 285: 2920029207.
Craig DB, Dombkowski AA. 2013. Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC Bioinformatics 14: 346.
Dombkowski AA. 2003. Disulfide by Design™: a computational method for the rational design of disulfide bonds in proteins. Bioinformatics 19: 1852-1853.
Dombkows ki A A, S ultana K Z, C raig DB. 2014. P rotein disulfide engineering. FEBS Lett. 588: 206-212.
Emanuelsson O, Brunak S, von Heijne G, Nielsen H. 2007. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2: 953-971.
Fu D, Huang H, Luo H, Wang Y, Yang P, Meng K, et al. 2008. A highly pH-stable phytase from Yersinia kristeensenii:cloning, expression, and characterization. Enzyme Microb. Technol. 42: 499-505.
Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, et al. 2004. Enhancing the thermal tolerance and gas tric p erformance of a m icrobial p hytas e for use as a phosphate-mobilizing monogastric-feed supplement. Appl. Environ. Microbiol. 70: 3041-3046.
Gu W-N, Huang H-Q, Yang P-L, Luo H-Y, Meng K, Wang Y-R, Yao B. 2007. Gene cloning, expression and characterization of a novel phytase from Hafnia alvei. Chin. J. Biotechnol. 23:1017-1021.
Guo C, Diao H, Lian Y, Yu H, Gao H, Zhang Y, Lin D. 2009. Recombinant expression and characterization of an epididymisspecific antimicrobial peptide BIN1b/SPAG11E. J. Biotechnol. 139: 33-37.
Guo C, Liu Y, Yu H, Du K, Gan Y, Huang H. 2016. A novel strategy for thermostability improvement of trypsin based on N-glycosylation within the Ω-loop region. J. Microbiol. Biotechnol. 26: 1163-1172.
Hatala JA, Detto M, Sonnentag O, Deverel SJ, Verfaillie J, Baldocchi DD. 2012. Greenhouse gas (CO2, CH4, H2O) fluxes from drained and flooded agricultural peatlands in the Sacramento-San Joaquin Delta. Agric. Ecosyst. Environ. 150: 1-18.
Hesampour A, Siadat SER, Malboobi MA, Mohandesi N, Arab SS, Ghahremanpour MM. 2015. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis. Appl. Biochem. Biotechnol. 175: 2528-2541.
Huang H, Luo H, Wang Y, Fu D, Shao N, Wang G, et al. 2008. A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Appl. Microbiol. Biotechnol. 80: 417-426.
Inoue H, Fujii T, Yoshimi M, Taylor LE II, Decker SR, Kishishita S, et al. 2013. Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. J. Ind. Microbiol. Biotechnol. 40: 823-830.
Jermutus L, Tessier M, Pasamontes L, van Loon A, Lehmann M. 2001. Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case. J. Biotechnol. 85: 15-24.
Kim M-S, Lei X. 2008. Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR. Appl. Microbiol. Biotechnol. 79: 69-75.
Knox SH, Sturtevant C, Matthes JH, Koteen L, Verfaillie J, Baldocchi D. 2015. Agricultural peatland restoration: effects of land-use change on greenhouse gas (CO2 and CH4) fluxes in the Sacramento-San Joaquin Delta. Global Change Biol. 21:750-765.
Le QAT, Joo JC, Yoo YJ, Kim YH. 2012. Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol. Bioeng. 109: 867-876.
Lei X-G, Weaver JD, Mullaney EJ, Ullah AH, Azain MJ. 2013. Phytase, a new life for an “old” enzyme. Annu. Rev. Anim. Biosci. 1: 283-309.
Liao Y, Li C-M, Chen H, Wu Q, Shan Z, Han X-Y. 2013. Site-directed mutagenesis improves the thermostability and catalytic efficiency of Aspergillus niger N25 phytase mutated by I44E and T252R. Appl. Biochem. Biotechnol. 171: 900-915.
Markowitz VM, Ivanova NN, Szeto E, Palaniappan K, Chu K, Dalevi D, et al. 2008. IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res. 36:D534-D538.
Markowitz VM, Mavromatis K, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. 2009. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 25: 2271-2278.
Merchant HA, McConnell EL, Liu F, Ramaswamy C, Kulkarni R P, B as it A W, M urdan S. 2 011. Ass es sment o f gastrointestinal pH, fluid and lymphoid tissue in the guinea pig, r abbit and pig, a nd implications for t heir us e in drug development. Eur. J. Pharm. Sci. 42: 3-10.
Miller RL, Fujii R. 2009. Plant community, primary productivity, and environmental conditions following wetland reestablishment in the Sacramento-San Joaquin Delta, California. Wetl. Ecol. Manag. 18: 1-16.
Nichols on FA, C hambers BJ, W illiams JR, U nwin R J. 1 999. Heavy metal contents of livestock feeds and animal manures in England and Wales. Bioresour. Technol. 70: 23-31.
Pandee P, Summpunn P, Wiyakrutta S, Isarangkul D, Meevootisom V. 2011. A thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris. J. Microbiol. 49: 257-264.
Patil KR, Roune L, McHardy AC. 2012. The PhyloPythiaS web server for taxonomic assignment of metagenome sequences. PLoS One 7: e38581.
Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Sanchez-Romero I, Ariza A, Wilson KS, Skjøt M, Vind J, De Maria L, et al. 2013. Mechanism of protein kinetic stabilization by engineered disulfide crosslinks. PLoS One 8: e70013.
Shivange AV, Dennig A, Schwaneberg U. 2014. Multi-site saturation by OmniChange yields a pH- and thermally improved phytase. J. Biotechnol. 170: 68-72.
Shivange AV, Serwe A, Dennig A, Roccatano D, Haefner S, Schwaneberg U. 2012. Directed evolution of a highly active Yersinia mollaretii phytase. Appl. Microbiol. Biotechnol. 95: 405-418.
Sone M, Kishigami S, Yoshihisa T, Ito K. 1997. Roles of disulfide bonds in bacterial alkaline phosphatase. J. Biol. Chem. 272: 6174-6178.
Tan H, Wu X, Xie L, Huang Z, Gan B, Peng W. 2015. Cloning, overexpression, and characterization of a metagenomederived phytase with optimal activity at low pH. J. Microbiol. Biotechnol. 25: 930-935.
Tan H, Wu X, Xie L, Huang Z, Peng W, Gan B. 2016. Identification and characterization of a mesophilic phytase highly resilient to high-temperatures from a fungus-garden associated metagenome. Appl. Microbiol. Biotechnol. 100: 22252241.
Tian YS, Peng RH, Xu J, Zhao W, Gao F, Fu XY, et al. 2011. Semi-rational site-directed mutagenesis of phyI1s from Aspergillus niger 113 at two residue to improve its phytase activity. Mol. Biol. Rep. 38: 977-982.
Ullah AHJ, Mullaney EJ. 1996. Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase. Biochem. Biophys. Res. Commun. 227: 311-317.
Vats P, Banerjee UC. 2005. Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem. J. Ind. Microbiol. Biotechnol. 32: 141-147.
Viader-Salvado JM, Gallegos-Lopez JA, Carreon-Trevino JG, Castillo-Galvan M, Rojo-Dominguez A, Guerrero-Olazaran M. 2010. Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris. Appl. Environ. Microbiol. 76: 6423-6430.
Vogt G, Argos P. 1997. Protein thermal stability: hydrogen bonds or internal packing? Fold Des. 2: S40-S46.
Wu T-H, Chen C-C, Cheng Y-S, Ko T-P, Lin C-Y, Lai H-L, et al. 2014. Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design. J. Biotechnol. 175: 1-6.
Yao MZ, Wang X, Wang W, Fu YJ, Liang AH. 2013. Improving the thermostability of Escherichia coli p hytas e, appA, by enhancement of glycosylation. Biotechnol. Lett. 35:1669-1676.
Zhang GQ, Dong XF, Wang ZH, Zhang Q, Wang HX, Tong JM. 2010. Purification, characterization, and cloning of a novel phytase with low pH optimum and strong proteolysis resistance from Aspergillus ficuum NTG-23. Bioresour. Technol. 101: 4125-4131.
Zhang WM, Mullaney EJ, Lei XG. 2007. Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase. Appl. Environ. Microbiol. 73:3069-3076.
Zhao QQ, Liu HL, Zhang Y, Zhang YZ. 2010. Engineering of protease-resistant phytase from Penicillium sp.: high thermal stability, low optimal temperature and pH. J. Biosci. Bioeng. 110: 638-645.
Zhu WH, Qiao DR, Huang M, Yang G, Xu H, Cao Y. 2010. Modifying thermostability of AppA from Escherichia coli. Curr. Microbiol. 61: 267-273.