Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

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

  • Tan, Hao (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Miao, Renyun (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Liu, Tianhai (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Cao, Xuelian (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Wu, Xiang (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Xie, Liyuan (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Huang, Zhongqian (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Peng, Weihong (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Gan, Bingcheng (Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences)
  • Received : 2016.04.19
  • Accepted : 2016.06.29
  • Published : 2016.10.28
Download PDF
⟨ Previous Next ⟩

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℃) 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℃ and 80℃, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.

Keywords

References

  1. Ariza A, Moroz OV, Blagova EV, Turkenburg JP, Waterman J, Roberts SM, et al. 2013. Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study. PLoS One 8: e65062. https://doi.org/10.1371/journal.pone.0065062
  2. Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201. https://doi.org/10.1093/bioinformatics/bti770
  3. 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. https://doi.org/10.1016/j.jbiotec.2008.08.011
  4. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. 2014. SWISS-MODEL: modelling protein tertiaryand quaternary structure using evolutionary information. Nucleic Acids Res. 42: W252-W258. https://doi.org/10.1093/nar/gku340
  5. 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. https://doi.org/10.1016/j.jcs.2013.10.003
  6. 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.
  7. 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: 29200-29207. https://doi.org/10.1074/jbc.M110.156596
  8. Craig DB, Dombkowski AA. 2013. Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins. BMC Bioinformatics 14: 346. https://doi.org/10.1186/1471-2105-14-346
  9. Dombkowski AA. 2003. Disulfide by DesignTM: a computational method for the rational design of disulfide bonds in proteins. Bioinformatics 19: 1852-1853. https://doi.org/10.1093/bioinformatics/btg231
  10. Dombkowski AA, Sultana KZ, Craig DB. 2014. Protein disulfide engineering. FEBS Lett. 588: 206-212. https://doi.org/10.1016/j.febslet.2013.11.024
  11. 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. https://doi.org/10.1038/nprot.2007.131
  12. 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. https://doi.org/10.1016/j.enzmictec.2008.01.014
  13. Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, et al. 2004. Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement. Appl. Environ. Microbiol. 70: 3041-3046. https://doi.org/10.1128/AEM.70.5.3041-3046.2004
  14. 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.
  15. 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. https://doi.org/10.1016/j.jbiotec.2008.10.003
  16. 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. https://doi.org/10.4014/jmb.1512.12070
  17. 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. https://doi.org/10.1016/j.agee.2012.01.009
  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. https://doi.org/10.1007/s12010-014-1440-y
  19. 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. https://doi.org/10.1007/s00253-008-1556-5
  20. 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. https://doi.org/10.1007/s10295-013-1286-2
  21. 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. https://doi.org/10.1016/S0168-1656(00)00373-4
  22. Kim M-S, Lei X. 2008. Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR. Appl. Microbiol. Biotechnol. 79: 69-75. https://doi.org/10.1007/s00253-008-1412-7
  23. 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. https://doi.org/10.1111/gcb.12745
  24. 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. https://doi.org/10.1002/bit.24371
  25. 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. https://doi.org/10.1146/annurev-animal-031412-103717
  26. 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. https://doi.org/10.1007/s12010-013-0380-2
  27. 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. https://doi.org/10.1093/nar/gkm869
  28. 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. https://doi.org/10.1093/bioinformatics/btp393
  29. Merchant HA, McConnell EL, Liu F, Ramaswamy C, Kulkarni RP, Basit AW, Murdan S. 2011. Assessment of gastrointestinal pH, fluid and lymphoid tissue in the guinea pig, rabbit and pig, and implications for their use in drug development. Eur. J. Pharm. Sci. 42: 3-10. https://doi.org/10.1016/j.ejps.2010.09.019
  30. Miller RL, Fujii R. 2009. Plant community, primary productivity, and environmental conditions following wetland re-establishment in the Sacramento-San Joaquin Delta, California. Wetl. Ecol. Manag. 18: 1-16. https://doi.org/10.1007/s11273-009-9143-9
  31. Nicholson FA, Chambers BJ, Williams JR, Unwin RJ. 1999. Heavy metal contents of livestock feeds and animal manures in England and Wales. Bioresour. Technol. 70: 23-31. https://doi.org/10.1016/S0960-8524(99)00017-6
  32. 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.
  33. Patil KR, Roune L, McHardy AC. 2012. The PhyloPythiaS web server for taxonomic assignment of metagenome sequences. PLoS One 7: e38581. https://doi.org/10.1371/journal.pone.0038581
  34. Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786. https://doi.org/10.1038/nmeth.1701
  35. 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. https://doi.org/10.1371/journal.pone.0070013
  36. Shivange AV, Dennig A, Schwaneberg U. 2014. Multi-site saturation by OmniChange yields a pH- and thermally improved phytase. J. Biotechnol. 170: 68-72. https://doi.org/10.1016/j.jbiotec.2013.11.014
  37. 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. https://doi.org/10.1007/s00253-011-3756-7
  38. Sone M, Kishigami S, Yoshihisa T, Ito K. 1997. Roles of disulfide bonds in bacterial alkaline phosphatase. J. Biol. Chem. 272: 6174-6178. https://doi.org/10.1074/jbc.272.10.6174
  39. Tan H, Wu X, Xie L, Huang Z, Gan B, Peng W. 2015. Cloning, overexpression, and characterization of a metagenome-derived phytase with optimal activity at low pH. J. Microbiol. Biotechnol. 25: 930-935. https://doi.org/10.4014/jmb.1411.11012
  40. 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: 2225-2241. https://doi.org/10.1007/s00253-015-7097-9
  41. 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. https://doi.org/10.1007/s11033-010-0192-1
  42. Ullah AHJ, Mullaney EJ. 1996. Disulfide bonds are necessary for structure and activity in Aspergillus ficuum phytase. Biochem. Biophys. Res. Commun. 227: 311-317. https://doi.org/10.1006/bbrc.1996.1506
  43. 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.
  44. 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. https://doi.org/10.1128/AEM.00253-10
  45. Vogt G, Argos P. 1997. Protein thermal stability: hydrogen bonds or internal packing? Fold Des. 2: S40-S46. https://doi.org/10.1016/S1359-0278(97)00062-X
  46. 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. https://doi.org/10.1016/j.jbiotec.2014.01.034
  47. 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. https://doi.org/10.1007/s10529-013-1255-x
  48. 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. https://doi.org/10.1016/j.biortech.2010.01.001
  49. 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. https://doi.org/10.1128/AEM.02970-06
  50. 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. https://doi.org/10.1016/j.jbiosc.2010.08.003
  51. 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. https://doi.org/10.1007/s00284-010-9606-5

Cited by

  1. Biochemical Characterization of a Psychrophilic Phytase from an Artificially Cultivable Morel Morchella importuna vol.27, pp.12, 2016, https://doi.org/10.4014/jmb.1708.08007
  2. ФИТАЗЫ И?ПЕРСПЕКТИВЫ ИХ ПРИМЕНЕНИЯ (ОБЗОР), "Прикладная биохимия и микробиология" vol.54, pp.4, 2016, https://doi.org/10.7868/s0555109918040025
  3. Review: Engineering of thermostable enzymes for industrial applications vol.2, pp.1, 2016, https://doi.org/10.1063/1.4997367
  4. Phytases and the Prospects for Their Application (Review) vol.54, pp.4, 2016, https://doi.org/10.1134/s0003683818040087
  5. Biochemical characterization of a key laccase-like multicopper oxidase of artificially cultivable Morchella importuna provides insights into plant-litter decomposition vol.9, pp.5, 2016, https://doi.org/10.1007/s13205-019-1688-6
  6. Rational Design of Alginate Lyase from Microbulbifer sp. Q7 to Improve Thermal Stability vol.17, pp.6, 2016, https://doi.org/10.3390/md17060378
  7. Integrative Structural and Computational Biology of Phytases for the Animal Feed Industry vol.10, pp.8, 2016, https://doi.org/10.3390/catal10080844