Cloning, Overexpression, and Characterization of a Metagenome-Derived Phytase with Optimal Activity at Low pH

Hao  Tan; Xiang  Wu; Liyuan  Xie; Zhongqian  Huang; Bingcheng  Gan; Weihong  Peng

doi:10.4014/jmb.1411.11012

Articles Service

Metrics

CrossRef [4]
View[582]
Download[257]

Note

Environmental Microbiology and Engineering

Cloning, Overexpression, and Characterization of a Metagenome-Derived Phytase with Optimal Activity at Low pH

Hao Tan ^{1, 2}, Xiang Wu ^{1, 2}, Liyuan Xie ^{1, 2}, Zhongqian Huang ^{1, 2}, Bingcheng Gan ^{1, 2} and Weihong Peng ^{1, 2*}

¹Institute of Soil and Fertilizer, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. China, ²R&D Department, JINDI Agricultural Biotechnology Inc., Chengdu, P.R. China

Received: November 7, 2014; Accepted: January 8, 2015

J. Microbiol. Biotechnol. 2015; 25(6): 930-935

Published June 28, 2015 https://doi.org/10.4014/jmb.1411.11012

Abstract

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 μmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37°C. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

Keywords

phytic acid, phytase, metagenome, pH-optimum, acidophilic

References

Barabote RD, Xie G, Brettin TS, Hinrichs SH, Fey PD, Jay JJ, et al. 2009. Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00. PLoS One 4: e7041.
Bilik K, Strzetelski J, Furgal-Dierzuk I, Sliwinski B. 2014. Effect of supplementing TMR diets with artificial saliva and acid buf on optimizing ruminal pH and fermentation activity in cows. Ann. Anim. Sci. 14: 585-593.
Böhm K, Herter T, Müller JJ, Borriss R, Heinemann U. 2010. Crystal structure of Klebsiella sp. ASR1 phytase suggests substrate binding to a preformed active site that meets the requirements of a plant rhizosphere enzyme. FEBS J. 277:1284-1296.
Ding Q, Yang P, Huang H, Wang Y, Yao B. 2010. Development status and research trends of phytases. J. Agric. Sci. Technol. 12: 27-33.
Dono ND, Sparks NH, Olukosi OA. 2014. Association between digesta pH, body weight, and nutrient utilization in chickens of different body weights and at different ages. J. Poult. Sci. 51: 180-184.
Em anuelsson O, B runak S, v on H eijne G, N ielsen H . 2007. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2: 953-971.
Fu D, Huang H, Meng K, Wang Y, Luo H, Yang P, et al. 2009. Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution. Biotechnol. Bioeng. 103:857-864.
Golovan S, Wang G, Zhang J, Forsberg CW. 2000. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can. J. Microbiol. 46:59-71.
Greiner R, da Silva LG, Couri S. 2009. Purification and characterization of an extracellular phytase from Aspergillus niger 11T53A9. Braz. J. Microbiol. 40: 795-807.
Hanson BT, Yagi JM, Jeon CO, Madsen EM. 2012. Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments. Environ. Microbiol. 14: 1544-1557.
Kostrewa D, Wyss M, D’Arcy A, Van Loon APGM. 1999. Crystal structure of Aspergillus niger pH 2.5 acid phosphatase at 2.4 Å resolution. J. Mol. Biol. 288: 965-974.
Lei X-G, Porres JM. 2003. Phytase enzymology, applications, and biotechnology. Biotechnol. Lett. 25: 1787-1794.
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 I-MA, Chu K, Kyrpides NC. 2009. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 25: 2271-2278.
Moesseler A, Kottendorf S, Liesner VG, Kamphues J. 2010. Impact of diets’ physical form (particle size; meal/pelleted) on the stomach content (dry matter content, pH, chloride concentration) of pigs. Livestock Sci. 134: 146-148.
Moesseler A, Wintermann M, Sander SJ, Kamphues J. 2012. Effect of diet grinding and pelleting fed either dry or liquid feed on dry matter and pH in the stomach of pigs and the development of gastric ulcers. J. Anim. Sci. 90: 343-345.
Mohammed R, Hunerberg M, McAllister TA, Beauchemin KA. 2014. Characterization of ruminal temperature and its relationship with ruminal pH in beef heifers fed growing and finishing diets. J. Anim. Sci. 92: 4650-4660.
Mukhametzyanova AD, Akhmetova AI, Sharipova MR. 2012. Microorganisms as phytase producers. Microbiology 81:267-275.
Mullaney EJ, Ullah AH. 2003. The term phytase comprises several different classes of enzymes. Biochem. Biophys. Res. Commun. 312: 179-184.
Olukosi OA, Dono ND. 2014. Modification of digesta pH and intestinal morphology with the use of benzoic acid or phytobiotics and the effects on broiler chicken growth performance and energy and nutrient utilization. J. Anim. Sci. 92: 3945-3953.
Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Qadis AQ, Goya S, Ikuta K, Yatsu M, Kimura A, Nakanishi S, Sato S. 2014. Effects of a bacteria-based probiotic on ruminal pH, volatile fatty acids and bacterial flora of Holstein calves. J. Vet. Med. Sci. 76: 877-885.
Reddy NR, Sathe SK, Salunkhe DK. 1982. Phytates in legumes and cereals. Adv. Food Res. 28: 1-92.
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.
Tomschy A, Brugger R, Lehmann M, Svendsen A, Vogel K, Kostrewa D, et al. 2002. Engineering of phytase for improved activity at low pH. Appl. Environ. Microbiol. 68: 1907-1913.
Ullah AH, Cummins BJ. 1987. Purification, N-terminal amino acid sequence and characterization of the pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum. Prep. Biochem. 17: 397-422.
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.
Wyss M, Pasamontes L, Remy R, Kohler J, Kusznir E, Gadient M, et al. 1998. Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger pH 2.5 acid phosphatase. Appl. Environ. Microbiol. 64: 4446-4451.
Yao MZ, Wang X, Wang W, Fu YJ, Liang AH. 2013. Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation. Biotechnol. Lett. 35:1669-1676.
Yao MZ, Zhang YH, Lu WL, Hu MQ, Wang W, Liang AH. 2012. Phytases: crystal structures, protein engineering and potential biotechnological applications. J. Appl. Microbiol. 112: 1-14.
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: 4125-4134.

Article Tools

Article

Note

J. Microbiol. Biotechnol. 2015; 25(6): 930-935

Published online June 28, 2015 https://doi.org/10.4014/jmb.1411.11012

Cloning, Overexpression, and Characterization of a Metagenome-Derived Phytase with Optimal Activity at Low pH

Hao Tan ^{1, 2}, Xiang Wu ^{1, 2}, Liyuan Xie ^{1, 2}, Zhongqian Huang ^{1, 2}, Bingcheng Gan ^{1, 2} and Weihong Peng ^{1, 2*}

¹Institute of Soil and Fertilizer, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R. China, ²R&D Department, JINDI Agricultural Biotechnology Inc., Chengdu, P.R. China

Received: November 7, 2014; Accepted: January 8, 2015

Abstract

A phytase gene was identified in a publicly available metagenome derived from subsurface
groundwater, which was deduced to encode for a protein of the histidine acid phosphatase
(HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and
cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the
recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 μmol P/min/mg, at
the pH optimum of 2.0 with the temperature of 37°C. Interestingly, the pH optimum of this
phytase is much lower in comparison with most HAP phytases known to date. It suggests that
the phytase could possess improved adaptability to the low pH condition caused by the
gastric acid in livestock and poultry stomachs.

Keywords: phytic acid, phytase, metagenome, pH-optimum, acidophilic

Abstract

References

Barabote RD, Xie G, Brettin TS, Hinrichs SH, Fey PD, Jay JJ, et al. 2009. Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00. PLoS One 4: e7041.
Bilik K, Strzetelski J, Furgal-Dierzuk I, Sliwinski B. 2014. Effect of supplementing TMR diets with artificial saliva and acid buf on optimizing ruminal pH and fermentation activity in cows. Ann. Anim. Sci. 14: 585-593.
Böhm K, Herter T, Müller JJ, Borriss R, Heinemann U. 2010. Crystal structure of Klebsiella sp. ASR1 phytase suggests substrate binding to a preformed active site that meets the requirements of a plant rhizosphere enzyme. FEBS J. 277:1284-1296.
Ding Q, Yang P, Huang H, Wang Y, Yao B. 2010. Development status and research trends of phytases. J. Agric. Sci. Technol. 12: 27-33.
Dono ND, Sparks NH, Olukosi OA. 2014. Association between digesta pH, body weight, and nutrient utilization in chickens of different body weights and at different ages. J. Poult. Sci. 51: 180-184.
Em anuelsson O, B runak S, v on H eijne G, N ielsen H . 2007. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2: 953-971.
Fu D, Huang H, Meng K, Wang Y, Luo H, Yang P, et al. 2009. Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution. Biotechnol. Bioeng. 103:857-864.
Golovan S, Wang G, Zhang J, Forsberg CW. 2000. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can. J. Microbiol. 46:59-71.
Greiner R, da Silva LG, Couri S. 2009. Purification and characterization of an extracellular phytase from Aspergillus niger 11T53A9. Braz. J. Microbiol. 40: 795-807.
Hanson BT, Yagi JM, Jeon CO, Madsen EM. 2012. Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments. Environ. Microbiol. 14: 1544-1557.
Kostrewa D, Wyss M, D’Arcy A, Van Loon APGM. 1999. Crystal structure of Aspergillus niger pH 2.5 acid phosphatase at 2.4 Å resolution. J. Mol. Biol. 288: 965-974.
Lei X-G, Porres JM. 2003. Phytase enzymology, applications, and biotechnology. Biotechnol. Lett. 25: 1787-1794.
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 I-MA, Chu K, Kyrpides NC. 2009. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 25: 2271-2278.
Moesseler A, Kottendorf S, Liesner VG, Kamphues J. 2010. Impact of diets’ physical form (particle size; meal/pelleted) on the stomach content (dry matter content, pH, chloride concentration) of pigs. Livestock Sci. 134: 146-148.
Moesseler A, Wintermann M, Sander SJ, Kamphues J. 2012. Effect of diet grinding and pelleting fed either dry or liquid feed on dry matter and pH in the stomach of pigs and the development of gastric ulcers. J. Anim. Sci. 90: 343-345.
Mohammed R, Hunerberg M, McAllister TA, Beauchemin KA. 2014. Characterization of ruminal temperature and its relationship with ruminal pH in beef heifers fed growing and finishing diets. J. Anim. Sci. 92: 4650-4660.
Mukhametzyanova AD, Akhmetova AI, Sharipova MR. 2012. Microorganisms as phytase producers. Microbiology 81:267-275.
Mullaney EJ, Ullah AH. 2003. The term phytase comprises several different classes of enzymes. Biochem. Biophys. Res. Commun. 312: 179-184.
Olukosi OA, Dono ND. 2014. Modification of digesta pH and intestinal morphology with the use of benzoic acid or phytobiotics and the effects on broiler chicken growth performance and energy and nutrient utilization. J. Anim. Sci. 92: 3945-3953.
Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Qadis AQ, Goya S, Ikuta K, Yatsu M, Kimura A, Nakanishi S, Sato S. 2014. Effects of a bacteria-based probiotic on ruminal pH, volatile fatty acids and bacterial flora of Holstein calves. J. Vet. Med. Sci. 76: 877-885.
Reddy NR, Sathe SK, Salunkhe DK. 1982. Phytates in legumes and cereals. Adv. Food Res. 28: 1-92.
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.
Tomschy A, Brugger R, Lehmann M, Svendsen A, Vogel K, Kostrewa D, et al. 2002. Engineering of phytase for improved activity at low pH. Appl. Environ. Microbiol. 68: 1907-1913.
Ullah AH, Cummins BJ. 1987. Purification, N-terminal amino acid sequence and characterization of the pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum. Prep. Biochem. 17: 397-422.
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.
Wyss M, Pasamontes L, Remy R, Kohler J, Kusznir E, Gadient M, et al. 1998. Comparison of the thermostability properties of three acid phosphatases from molds: Aspergillus fumigatus phytase, A. niger phytase, and A. niger pH 2.5 acid phosphatase. Appl. Environ. Microbiol. 64: 4446-4451.
Yao MZ, Wang X, Wang W, Fu YJ, Liang AH. 2013. Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation. Biotechnol. Lett. 35:1669-1676.
Yao MZ, Zhang YH, Lu WL, Hu MQ, Wang W, Liang AH. 2012. Phytases: crystal structures, protein engineering and potential biotechnological applications. J. Appl. Microbiol. 112: 1-14.
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: 4125-4134.