Journal of Microbiology and Biotechnology

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

DOI QR Code

Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters

  • Li, Jiadi (Dalian Biocatalytic Engineering Laboratory, School of Biological Engineering, Dalian Polytechnic University) ;
  • Li, Xinli (Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) ;
  • Gai, Yuanming (Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences) ;
  • Sun, Yumei (Dalian Biocatalytic Engineering Laboratory, School of Biological Engineering, Dalian Polytechnic University) ;
  • Zhang, Dawei (Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences)
  • Received : 2018.11.12
  • Accepted : 2019.02.06
  • Published : 2019.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric acid, and have been utilized to increase the availability of nutrients in animal feed and mitigate environmental pollution. However, the enzymes' low thermostability has limited their application during the feed palletization process. In this study, a combination of B-value calculation and protein surface engineering was applied to rationally evolve the heat stability of Escherichia coli phytase. After systematic alignment and mining for homologs of the original phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were chosen and used to identify the B-values and spatial distribution of key amino acid residues. Consequently, thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries. After five rounds of iterative mutation screening, the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type, the residual enzyme activity of the mutant increased from 20% to 75% after incubation at $90^{\circ}C$ for 5 min. Compared with traditional methods, the rational engineering approach used in this study reduces the screening workload and provides a reference for future applications of phytases as green catalysts.

Keywords

References

  1. Lei XG, Weaver JD, Mullaney E, 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
  2. Yin HF, Fan BL, Yang B, Liu YF, Luo J, Tian XH, et al. 2006. Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control. J. Animal Sci. 84: 513-519. https://doi.org/10.2527/2006.843513x
  3. Lei XG, Porres JM, Mullaney EJ, Brinchpedersen H. 2007. Phytase: Source, Structure and Application, pp. 505-529. In Polaina J, MacCabe AP (eds.), Industrial enzymes: Structure, function and applications, Ed. Springer, New York.
  4. Reetz MT, Peyralans JJ, Maichele A, Fu Y, Maywald M. 2006. Directed evolution of hybrid enzymes: Evolving enantioselectivity of an achiral Rh-complex anchored to a protein. Chem. Commun. 41: 4318-4320.
  5. Herger M, van Roye P, Romney DK, Brinkmann-Chen S, Buller AR, Arnold FH. 2016. Synthesis of beta-branched tryptophan analogues using an engineered subunit of tryptophan synthase. J. Am. Chem. Soc. 138: 8388-8391. https://doi.org/10.1021/jacs.6b04836
  6. Gabriel J. Rocklin TMC, Inna Goreshnik, Alex Ford, Scott Houliston, et al. 2017. Global analysis of protein folding using massively parallel design, synthesis, and testing. Science 357: 168-175. https://doi.org/10.1126/science.aan0693
  7. Reetz MT, Soni P, Fernandez L, Gumulya Y, Carballeira JD. 2010. Increasing the stability of an enzyme toward hostile organic solvents by directed evolution based on iterative saturation mutagenesis using the B-FIT method. Chem. Commun (Camb). 46: 8657-8658. https://doi.org/10.1039/c0cc02657c
  8. Acevedo JP, Reetaz MT, Asenjo JA, Parra LP. 2017. One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase. Enzyme Microb. Technol. 100: 60-70. https://doi.org/10.1016/j.enzmictec.2017.02.005
  9. Stemmer WP. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91: 10747-10751. https://doi.org/10.1073/pnas.91.22.10747
  10. Chen K, Arnold FH. 1993. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc. Natl. Acad. Sci. USA 90: 5618-5622. https://doi.org/10.1073/pnas.90.12.5618
  11. Shivange AV, Roccatano D, Schwaneberg U. 2016. Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution. Appl. Microbiol. Biotechnol. 100: 227-242. https://doi.org/10.1007/s00253-015-6959-5
  12. Mootapally CS, Nathani NM, Patel AK, Jakhesara SJ, Joshi CG. 2016. Mining of ruminant microbial phytase (RPHY1) from metagenomic data of mehsani buffalo breed: identification, gene cloning, and characterization. J. Mol. Microbiol. Biotechnol. 26: 252-260. https://doi.org/10.1159/000445321
  13. Mittal A, Singh G, Goyal V, Yadav A. 2011. Isolation and biochemical characterization of acido-thermophilic extracellular phytase producing bacterial strain for potential application in poultry feed. Jundishapur. J. Microbiol. 4: 273-282.
  14. Singh B, Satyanarayana T. 2011. Phytases from thermophilic molds: Their production, characteristics and multifarious applications. Process Biochem. 46: 1391-1398. https://doi.org/10.1016/j.procbio.2011.03.009
  15. Hesampour A, Siadat SE, 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: 25-28. https://doi.org/10.1007/s12010-014-1243-1
  16. Xin GL, Porres JM. 2003. Phytase enzymology, applications, and biotechnology. Biotechnol. Lett. 25: 1787-1794. https://doi.org/10.1023/A:1026224101580
  17. 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
  18. Luo H, Huang H, Yang P, Wang Y, Yuan T, Wu N, et al. 2007. A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: gene cloning and overexpression in Pichia pastoris. Curr. Microbiol. 55: 185-192. https://doi.org/10.1007/s00284-006-0586-4
  19. Fei B, Xu H, Cao Y, Ma S, Guo H, Song T, et al. 2013. A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase. J. Ind. Microbiol. Biotechnol. 40: 457-464. https://doi.org/10.1007/s10295-013-1260-z
  20. Shivange AV, Schwaneberg U, Roccatano D. 2010. Conformational dynamics of active site loop in Escherichia coli phytase. Biopolymers 93: 994-1002. https://doi.org/10.1002/bip.21513
  21. Noorbatcha IA, Sultan AM, Salleh HM, Amid A. 2013. Understanding thermostability factors of Aspergillus niger PhyA phytase: a molecular dynamics study. Protein J. 32: 309-316. https://doi.org/10.1007/s10930-013-9489-y
  22. Fei B, Cao Y, Xu H, Li X, Song T, Fei Z, et al. 2013. AppA Cterminal plays an important role in its thermostability in Escherichia coli. Curr. Microbiol. 66: 374-378. https://doi.org/10.1007/s00284-012-0283-4
  23. Fei B, Xu H, Zhang F, Li X, Ma S, Cao Y, et al. 2013. Relationship between Escherichia coli AppA phytase's thermostability and salt bridges. J. Biosci. Bioeng. 115: 623-627. https://doi.org/10.1016/j.jbiosc.2012.12.010
  24. Berkmen M, Boyd D, Beckwith J. 2005. The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase. J. Biol. Chem. 280: 11387-11394. https://doi.org/10.1074/jbc.M411774200
  25. Wu TH, Chen CC, Cheng YS, Ko TP, Lin CY, Lai HL, 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
  26. Haiquan Yang, Xinyao Lu, Long Liu, Jianghua Li, Hyundong Shin, et al. 2013. Fusion of an oligopeptide to the N terminus of an alkaline ${\alpha}$-amylase from Alkalimonas amylolytica simultaneously improves the enzyme's catalytic efficiency, thermal stability, and resistance to oxidation. Appl. Environ. Microbiol. 79: 3049-3058. https://doi.org/10.1128/AEM.03785-12
  27. M.R.N.Murthy SP. 2000. Protein thermal stability: insights from atomic displacement parameters (B values). Protein Eng. 13: 9-13. https://doi.org/10.1093/protein/13.1.9
  28. Reetz MT, Carballeira JD, Vogel A. 2006. Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew. Chem. Int. Ed. Engl. 45: 7745-7751. https://doi.org/10.1002/anie.200602795
  29. Sutiono S, Carsten J, Sieber V. 2018. Structure-guided engineering of alpha-keto acid decarboxylase for the production of higher alcohols at elevated temperature. ChemSusChem. 11: 3334-3344.
  30. Larkin MA, Blackshields G, Brown NP, Chenna R, Mcgettigan PA, Mcwilliam H, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948. https://doi.org/10.1093/bioinformatics/btm404
  31. Studier FW. 2005. Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41: 207-234. https://doi.org/10.1016/j.pep.2005.01.016
  32. Liu ZQ, Mahmood T, Yang PC. 2012. Western blot: technique, theory and trouble shooting. N. Am. J. Med. Sci. 4: 429-434. https://doi.org/10.4103/1947-2714.100998
  33. Yin QQ, Zheng QH, Kang XT. 2007. Biochemical characteristics of phytases from fungi and the transformed microorganism. Anim. Feed Sci. Technol. 132: 341-350. https://doi.org/10.1016/j.anifeedsci.2006.03.016
  34. Gooch JW. 2011. Lineweaver-Burk Plot. pp. 904-904. Encyclopedic Dictionary of Polymers. Ed. Springer New York.
  35. Lim D, Golovan S, Forsberg CW, Jia Z. 2000. Crystal structures of Escherichia coli phytase and its complex with phytate. Nat. Struct. Biol. 7: 108-113. https://doi.org/10.1038/72371
  36. Martin A, Schmid FSV. 2001. In-vitro selection of highly stabilized protein variants with optimized surface. J. Mol. Biol. 309: 717-726. https://doi.org/10.1006/jmbi.2001.4698
  37. Alsop E, Silver M, Livesay DR. 2003. Optimized electrostatic surfaces parallel increased thermostability: a structural bioinformatic analysis. Protein Eng. 16: 871-874. https://doi.org/10.1093/protein/gzg131
  38. Reetz MT, Carballeira JD. 2007. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2: 891-903. https://doi.org/10.1038/nprot.2007.72
  39. Quezada AG, Diaz-Salazar AJ, Cabrera N, Perez-Montfort R, Pineiro A, Costas M. 2017. Interplay between protein thermal flexibility and kinetic stability. Structure 25: 167-179. https://doi.org/10.1016/j.str.2016.11.018
  40. Radivojac P, Obradovic Z, Smith DK, Zhu G, Vucetic S, Brown CJ, et al. 2004. Protein flexibility and intrinsic disorder. Protein Soc. 13: 71-80. https://doi.org/10.1110/ps.03128904
  41. Menendezarias L, Argos P. 1989. Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. J. Mol. Biol. 206: 397-406. https://doi.org/10.1016/0022-2836(89)90488-9
  42. Xiao S, Patsalo V, Shan B, Bi Y, Green DF, Raleigh DP. 2013. Rational modification of protein stability by targeting surface sites leads to complicated results. Proc. Natl. Acad. Sci. USA 110: 11337-11342. https://doi.org/10.1073/pnas.1222245110
  43. Vogt G, Argos P. 1997. Protein thermal stability: hydrogen bonds or internal packing? Folding Design. 2: S40-S46. https://doi.org/10.1016/S1359-0278(97)00062-X
  44. B Garrett J, A Kretz K, O'Donoghue E, Kerovuo J, Kim W, R Barton N, 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

Cited by

  1. Integrative Structural and Computational Biology of Phytases for the Animal Feed Industry vol.10, pp.8, 2019, https://doi.org/10.3390/catal10080844