Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Biochemical Characterization of a Psychrophilic Phytase from an Artificially Cultivable Morel Morchella importuna

  • Tan, Hao (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Tang, Jie (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Li, Xiaolin (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Liu, Tianhai (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Miao, Renyun (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Huang, Zhongqian (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Wang, Yong (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Gan, Bingcheng (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences) ;
  • Peng, Weihong (National-Local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences)
  • Received : 2017.08.04
  • Accepted : 2017.09.15
  • Published : 2017.12.28
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Abstract

Psychrophilic phytases suitable for aquaculture are rare. In this study, a phytase of the histidine acid phosphatase (HAP) family was identified in Morchella importuna, a psychrophilic mushroom. The phytase showed 38% identity with Aspergillus niger PhyB, which was the closest hit. The M. importuna phytase was overexpressed in Pichia pastoris, purified, and characterized. The phytase had an optimum temperature at $25^{\circ}C$, which is the lowest among all the known phytases to our best knowledge. The optimum pH (6.5) is higher than most of the known HAP phytases, which is fit for the weak acidic condition in fish gut. At the optimum pH and temperature, MiPhyA showed the maximum activity level ($2,384.6{\pm}90.4{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}$, suggesting that the enzyme possesses a higher activity level over many known phytases at low temperatures. The phytate-degrading efficacy was tested on three common feed materials (soybean meal/rapeseed meal/corn meal) and was compared with the well-known phytases of Escherichia coli and A. niger. When using the same amount of activity units, MiPhyA could yield at least $3{\times}$ more inorganic phosphate than the two reference phytases. When using the same weight of protein, MiPhyA could yield at least $5{\times}$ more inorganic phosphate than the other two. Since it could degrade phytate in feed materials efficiently under low temperature and weak acidic conditions, which are common for aquacultural application, MiPhyA might be a promising candidate as a feed additive enzyme.

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References

  1. Gulati HK, Chadha BS, Saini HS. 2007. Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7. Acta Microbiol. Immunol. Hung. 54: 121-138. https://doi.org/10.1556/AMicr.54.2007.2.3
  2. Borgi M, Boudebbouze S, Aghajari N, Szukala F, Pons N, Maguin E, et al. 2014. The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization. Appl. Microbiol. Biotechnol. 98: 5937-5947. https://doi.org/10.1007/s00253-013-5421-9
  3. Tan H, W u X, Xie L, H uang Z, G an 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
  4. Cao L, Wang W, Yang C, Yang Y, Diana J, Yakupitiyage A, et al. 2007. Application of microbial phytase in fish feed. Enzyme Microb. Technol. 40: 497-507. https://doi.org/10.1016/j.enzmictec.2007.01.007
  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. https://doi.org/10.1159/000446567
  6. Ushasree MV, Shyam K, Vidya J, Pandey A. 2017. Microbial phytase: impact of advances in genetic engineering in revolutionizing its properties and applications. Bioresour. Technol. 245: 1790-1799. https://doi.org/10.1016/j.biortech.2017.05.060
  7. Tran TT, Mamo G, Mattiasson B, Hatti-Kaul R. 2010. A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli. J. Ind. Microbiol. Biotechnol. 37: 279-287. https://doi.org/10.1007/s10295-009-0671-3
  8. 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
  9. 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
  10. Rebello S, Jose L, Sindhu R, Aneesh EM. 2017. Molecular advancements in the development of thermostable phytases. Appl. Microbiol. Biotechnol. 101: 2677-2689. https://doi.org/10.1007/s00253-017-8195-7
  11. Huang H, Luo H, Wang Y, Fu D, Shao N, Yang P, et al. 2009. Novel low-temperature-active phytase from Erwinia carotovora var. carotovota ACCC 10276. J. Microbiol. Biotechnol. 19: 1085-1091.
  12. Huang H, Shao N, Wang Y, L u o H, Y ang P, Zhou Z, et al. 2009. A novel beta-propeller phytase from Pedobacter nyackensis MJ11 CGMCC 2503 with potential as an aquatic feed additive. Appl. Microbiol. Biotechnol. 83: 249-259. https://doi.org/10.1007/s00253-008-1835-1
  13. Yu P, Wang X-T, Liu J-W. 2015. Purification and characterization of a novel cold-adapted phytase from Rhodotorula mucilaginosa strain JMUY14 isolated from Antarctic. J. Basic Microbiol. 55: 1029-1039. https://doi.org/10.1002/jobm.201400865
  14. Peng W, Tang J, He X, Chen Y, Tan H. 2016. Status analysis of morel artificial cultivation in Sichuan. Edible Med. Mushrooms 24: 145-150.
  15. Schmidt EL. 1983. Spore germination of and carbohydrate colonization by Morchella esculenta at different soil temperatures. Mycologia 75: 870-875. https://doi.org/10.2307/3792778
  16. Xu H, Sun L-P, Shi Y-Z, Wu Y-H, Zhang B, Zhao D-Q. 2008. Optimization of cultivation conditions for extracellular polysaccharide and mycelium biomass by Morchella esculenta As51620. Biochem. Eng. J. 39: 66-73. https://doi.org/10.1016/j.bej.2007.08.013
  17. Goldway M, Amir R, Goldberg D, Hadar Y, Levanon D. 2000. Morchella conica exhibiting a long fruiting season. Mycol. Res. 104: 1000-1004. https://doi.org/10.1017/S0953756200002598
  18. Hong DK, Jang S-H, Lee C. 2017. Gene cloning and characterization of a psychrophilic phthalate esterase with organic solvent tolerance from an Arctic bacterium Sphingomonas glacialis PAMC 26605. J. Mol. Catal. B Enzyme. 133: S337-S345.
  19. Feller G, Gerday C. 2003. Psychrophilic enzymes: hot topics in cold adaptation. Nat. Rev. Microbiol. 1: 200-208. https://doi.org/10.1038/nrmicro773
  20. Feller G, Gerday C. 1997. Psychrophilic enzymes: molecular basis of cold adaptation. Cell. Mol. Life Sci. 53: 830-841. https://doi.org/10.1007/s000180050103
  21. Parvizpour S, Razmara J, Ramli ANM, Md Illias R, Shamsir MS. 2014. Structural and functional analysis of a novel psychrophilic $\beta$-mannanase from Glaciozyma antarctica PI12. J. Comput. Aided Mol. Des. 28: 685-698. https://doi.org/10.1007/s10822-014-9751-1
  22. 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
  23. Tan H, Barret M, Mooij MJ, Rice O, Morrissey JP, Dobson AD, et al. 2013. Long-term phosphorus fertilisation increased the diversity of the total bacterial community and the phoD phosphorus mineraliser group in pasture soils. Biol. Fertil. Soils 49: 661-672. https://doi.org/10.1007/s00374-012-0755-5
  24. Letunic I, Bork P. 2006. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23: 127-128.
  25. Fu D, Li Z, Huang H, Yuan T, Shi P, Luo H, et al. 2011. Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site. Appl. Microbiol. Biotechnol. 90: 1295-1302. https://doi.org/10.1007/s00253-011-3171-0
  26. Hu ang H, L u o H, W ang 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
  27. Tan H, Miao R, Liu T, Cao X, Wu X, Xie L, et al. 2016. Enhancing thermal resistance of a novel Acidobacteria-derived phytase by engineering of disulfide bridges. J. Microbiol. Biotechnol. 26: 1717-1722. https://doi.org/10.4014/jmb.1604.04051
  28. Bradford MM. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  29. 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
  30. Uchida H, Arakida S, Sakamoto T, Kawasaki H. 2006. Expression of Aspergillus oryzae phytase gene in Aspergillus oryzae RIB40 $niaD^-$. J. Biosci. Bioeng. 102: 564-567. https://doi.org/10.1263/jbb.102.564
  31. Heinonen JK, Lahti RJ. 1981. A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal. Biochem. 113: 313-317. https://doi.org/10.1016/0003-2697(81)90082-8
  32. Yao MZ, Zhang YH, Lu WL, Hu MQ, Wang W, L iang AH. 2012. Phytases: crystal structures, protein engineering and potential biotechnological applications. J. Appl. Microbiol. 112: 1-14. https://doi.org/10.1111/j.1365-2672.2011.05181.x
  33. Lassen SF, Breinholt J , Ostergaard P R, B ru gger R , Bischoff A, Wyss M, et al. 2001. Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens. Appl. Environ. Microbiol. 67: 4701-4707. https://doi.org/10.1128/AEM.67.10.4701-4707.2001
  34. Ushasree M, Vidya J, Pandey A. 2014. Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression. Biotechnol. Lett. 36: 85-91. https://doi.org/10.1007/s10529-013-1322-3
  35. Pasamontes L, Haiker M, Wyss M, Tessier M, van Loon AP. 1997. Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Appl. Environ. Microbiol. 63: 1696-1700.
  36. Berka RM, Rey MW, Brown KM, Byun T, Klotz AV. 1998. Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus. Appl. Environ. Microbiol. 64: 4423-4427.
  37. Tan H, Mooij MJ, Barret M, Hegarty PM, Harington C, Dobson AD, et al. 2014. Identification of novel phytase genes from an agricultural soil-derived metagenome. J. Microbiol. Biotechnol. 24: 113-118. https://doi.org/10.4014/jmb.1307.07007
  38. Niu C, Yang P, Luo H, Huang H, Wang Y, Yao B. 2017. Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency. Sci. Rep. 7: e42133. https://doi.org/10.1038/srep42133
  39. Shao N, Huang H, Meng K, Luo H, Wang Y, Yang P, et al. 2008. Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074. J. Microbiol. Biotechnol. 18: 1221-1226.
  40. 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
  41. Shi P, Huang H, Wang Y, Luo H, Wu B, Meng K, et al. 2008. A novel phytase gene appA from Buttiauxella sp. GC21 isolated from grass carp intestine. Aquaculture 275: 70-75. https://doi.org/10.1016/j.aquaculture.2008.01.021
  42. Hu ang H, Luo H, Yang P, Meng K, Wang Y, Yu an T , et al. 2006. A novel phytase with preferable characteristics from Yersinia intermedia. Biochem. Biophys. Res. Commun. 350: 884-889. https://doi.org/10.1016/j.bbrc.2006.09.118
  43. Vats P, Banerjee UC. 2005. Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phospho-hydrolase) from a hyper-producing strain of Aspergillus niger van Teighem. J. Ind. Microbiol. Biotechnol. 32: 141-147. https://doi.org/10.1007/s10295-005-0214-5
  44. Singh B, Satyanarayana T. 2009. Characterization of a HAP-phytase from a thermophilic mould Sporotrichum thermophile. Bioresour. Technol. 100: 2046-2051. https://doi.org/10.1016/j.biortech.2008.10.025
  45. Kim H-W, Kim Y-O, Lee J-H, Kim K-K, Kim Y-J. 2003. Isolation and characterization of a phytase with improved properties from Citrobacter braakii. Biotechnol. Lett. 25: 1231-1234. https://doi.org/10.1023/A:1025020309596
  46. Weaver JD, Ullah AHJ, Sethumadhavan K, Mullaney EJ, Lei XG. 2009. Impact of assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases. J. Agric. Food Chem. 57: 5315-5320. https://doi.org/10.1021/jf900261n
  47. Kumar V, Sinha AK, Makkar HPS, De Boeck G, Becker K. 2012. Phytate and phytase in fish nutrition. J. Anim. Physiol. Anim. Nutr. 96: 335-364. https://doi.org/10.1111/j.1439-0396.2011.01169.x
  48. Nwanna LC, Eisenreich R, Schwarz FJ. 2007. Effect of wet-incubation of dietary plant feedstuffs with phytases on growth and mineral digestibility by common carp (Cyprinus carpio L). Aquaculture 271: 461-468. https://doi.org/10.1016/j.aquaculture.2007.04.020
  49. Gonzalez de la Huebra MJ. 2005. Evaluation report on the analytical methods submitted in connection with the application for authorisation as a feed additive according to Regulation (EC) No 1831/2003: $Optiphos^{(R)}$. European Union Reference Laboratory for Feed Additives (EURL-FA), JRC Geel, Belgium.

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