Journal of Microbiology and Biotechnology

  • 제34권5호
  • /
  • Pages.1119-1125
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  • 2024
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Improved Thermal Stability of a Novel Acidophilic Phytase

  • Byung Sam Son (Institute of Biotechnology, CJ CheilJedang Co.) ;
  • So Hyeong Kim (Institute of Biotechnology, CJ CheilJedang Co.) ;
  • Hye-Young Sagong (Institute of Biotechnology, CJ CheilJedang Co.) ;
  • Su Rin Lee (Institute of Biotechnology, CJ CheilJedang Co.) ;
  • Eun Jung Choi (Institute of Biotechnology, CJ CheilJedang Co.)
  • 투고 : 2023.11.27
  • 심사 : 2024.03.24
  • 발행 : 2024.05.28
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초록

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris, TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70℃. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60℃. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80℃ heat treatment without significant loss of or with increased activity.

키워드

과제정보

Thanks to Hong Gon Kim for his experimental support.

참고문헌

  1. Hidvegi M, Lasztity R. 2002. Phytic acid content of cereals and legumes and interaction with proteins. Periodica Polytechnica Ser. Chem. Eng. 46: 56-94.
  2. Selle PH, Ravindran V. 2008. Phytate-degrading enzymes in pig nutrition. Livest. Sci. 113: 99-122.
  3. Silva VM, Putti FF, White PJ, Reis ARD. 2021. Phytic acid accumulation in plants: Biosynthesis pathway regulation and role in human diet. Plant Physiol Biochem. 164: 132-146.
  4. Hao T, Renyun M, Tianhai L, Xuelian C, Xiang W, Liyuan X, et al. 2016. Enhancing the thermal resistance of a novel acidobacteria-derived phytase by engineering of disulfide bridges. J. Microbiol. Biotechnol. 26: 1717-1722.
  5. Tan H, Wu X, Xie L, Huang Z, Peng W, Gan B. 2016. A novel phytase derived from an acidic peat-soil microbiome showing high stability under acidic plus pepsin conditions. J. Mol. Microbiol. Biotechnol. 26: 291-301.
  6. Kumar V, Sinha AK. 2018. Chapter 3 - General aspects of phytases, pp. 53-72. In Nunes CS, Kumar V (eds.), Enzymes in Human and Animal Nutrition, Ed. Academic Press.
  7. Mullaney EJ, Ullah AH. 2003. The term phytase comprises several different classes of enzymes. Biochem. Biophys. Res. Commun. 312: 179-184.
  8. Greiner R, Konietzny U. 2010. Phytases: Biochemistry, enzymology and characteristics relevant to animal feed use. Enzymes Farm Anim. Nutr. 2: 96-128.
  9. Inborr J, Bedford MR. 1994. Stability of feed enzymes to steam pelleting during feed processing. Anim. Feed Sci. Technol. 46: 179-196.
  10. Amerah AM, Ravindran V, Lentle RG, Thomas DG. 2008. Influence of feed particle size on the performance, energy utilization, digestive tract development, and digesta parameters of broiler starters fed wheat- and corn-based diets. Poult. Sci. 87: 2320-2328.
  11. Zhang GQ, Wu YY, Ng TB, Chen QJ, Wang HX. 2013. A phytase characterized by relatively high pH tolerance and thermostability from the shiitake mushroom Lentinus edodes. Biomed Res. Int. 2013: 540239.
  12. Kumar R. 2019. Simplified protocol for faster transformation of (a large number of) Pichia pastoris strains. Yeast 36: 399-410.
  13. Minamoto T. 2017. Random mutagenesis by error-prone polymerase chain reaction using a heavy water solvent. Methods Mol. Biol. 1498: 491-495.
  14. Ko J, Park H, Seok C. 2012. GalaxyTBM: template-based modeling by building a reliable core and refining unreliable local regions. BMC Bioinformatics 13: 198.
  15. Kunkel TA. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA82: 488-492.
  16. Bohm K, Herter T, Muller 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.
  17. 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.
  18. Oakley AJ. 2010. The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic. Biochem. Biophys. Res. Commun. 397: 745-749.
  19. Sanchez-Romero I, Ariza A, Wilson KS, Skjot M, Vind J, De Maria L, et al. 2013. Mechanism of protein kinetic stabilization by engineered disulfide crosslinks. PLoS One 8: e70013.
  20. Xiang T, Liu Q, Deacon AM, Koshy M, Kriksunov IA, Lei XG, et al. 2004. Crystal structure of a heat-resilient phytase from Aspergillus fumigatus, carrying a phosphorylated histidine. J. Mol. Biol. 339: 437-445.
  21. Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Deycke R, et al. 2010. The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb. Cell Fact. 9: 49.
  22. Dikbas N, Ucar S, Alim S. 2023. Purification of phytase enzyme from Lactobacillus brevis and biochemical properties. Biologia 78: 2583-2591.
  23. Mitchell DB, Vogel K, Weimann BJ, Pasamontes L, van Loon A. 1997. The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila. Microbiology (Reading) 143 (Pt 1): 245-252.
  24. Shi XW, Sun ML, Zhou B, Wang XY. 2009. Identification, characterization, and overexpression of a phytase with potential industrial interest. Can. J. Microbiol. 55: 599-604.
  25. 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.
  26. Wolde S, Negesse T, Melesse A. 2011. The effect of dietary protein concentration on nutrient utilization of Rhode Island Red chicken in Wolaita (Southern Ethiopia). Trop. Subtrop. Agroecosystems 14: 271-278.
  27. Deka G, Dutta H, Sharma P. 2023. review: hydrothermal processing of millets and effect on techno-functional and nutritional propertieg. Food Process Eng. 46: e14408.
  28. Han C, Wang Q, Sun Y, Yang R, Liu M, Wang S, et al. 2020. Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites. Biotechnol. Biofuels 13: 30.
  29. Yang M-J, Lee HW, Kim H. 2017. Enhancement of thermostability of Bacillus subtilis endoglucanase by error-prone PCR and DNA shuffling. Appl. Biol. Chem. 60: 73-78.
  30. Si M, Xu Q, Jiang L, Huang H. 2016. SpyTag/SpyCatcher cyclization enhances the thermostability of firefly luciferase. PLoS One 11: e0162318.
  31. Nemethy G, Leach SJ, Scheraga HA. 1966. The influence of amino acid side chains on the free energy of Helix-Coil transitions1. J. Phys. Chem. 70: 998-1004.
  32. Watanabe K, Suzuki Y. 1998. Protein thermostabilization by proline substitutions. J. Mol. Catalysis B: Enzymatic. 4: 167-180.
  33. Bashirova A, Pramanik S, Volkov P, Rozhkova A, Nemashkalov V, Zorov I, et al. 2019. Disulfide bond engineering of an endoglucanase from Penicillium verruculosum to improve its thermostability. Int. J. Mol. Sci. 20: 1602.
  34. Li G, Fang X, Su F, Chen Y, Xu L, Yan Y. 2018. Enhancing the thermostability of Rhizomucor miehei lipase with a limited screening library by rational-design point mutations and disulfide bonds. Appl. Environ. Microbiol. 84: e02129-17.
  35. Li L, Zhang S, Wu W, Guan W, Deng Z, Qiao H. 2019. Enhancing thermostability of Yarrowia lipolytica lipase 2 through engineering multiple disulfide bonds and mitigating reduced lipase production associated with disulfide bonds. Enzyme Microb. Technol. 126: 41-49.
  36. Zhu W, Qin L, Xu Y, Lu H, Wu Q, Li W, et al. 2023. Three molecular modification strategies to improve the thermostability of xylanase XynA from Streptomyces rameus L2001. Foods 12: 879.
  37. Navone L, Vogl T, Luangthongkam P, Blinco JA, Luna-Flores CH, Chen X, et al. 2021. Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris. Biotechnol. Biofuels 14: 80.
  38. Gao X, Dong X, Li X, Liu Z, Liu H. 2020. Prediction of disulfide bond engineering sites using a machine learning method. Sci. Rep. 10: 10330.
  39. Chen CC, Cheng KJ, Ko TP, Guo RT. 2015. Current progresses in phytase research: Three-dimensional structure and protein engineering. ChemBioEng. Rev. 2: 76-86.
  40. Bei J, Chen Z, Fu J, Jiang Z, Wang J, Wang X. 2009. Structure-based fragment shuffling of two fungal phytases for combination of desirable properties. J. Biotechnol. 139: 186-193.
  41. Yang H, Qu J, Zou W, Shen W, Chen X. 2021. An overview and future prospects of recombinant protein production in Bacillus subtilis. Appl. Microbiol. Biotechnol. 105: 6607-6626.
  42. Li Y-y, Zhong K-x, Hu A-h, Liu D-n, Chen L-z, Xu S-d. 2015. High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris. Protein Expr. Purif. 108: 90-96.
  43. Yang X, Cong H, Song J, Zhang J. 2013. Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris. World J. Microbiol. Biotechnol. 29: 2087-2094.
  44. Kiiskinen LL, Kruus K, Bailey M, Ylosmaki E, Siika-Aho M, Saloheimo M. 2004. Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology (Reading) 150: 3065-3074.