Identification and characterization of a mesophilic phytase highly resilient to high-temperatures from a fungus-garden associated metagenome
- 437 Downloads
Phytases are enzymes degrading phytic acid and thereby releasing inorganic phosphate. While the phytases reported to date are majorly from culturable microorganisms, the fast-growing quantity of publicly available metagenomic data generated in the last decade has enabled bioinformatic mining of phytases in numerous data mines derived from a variety of ecosystems throughout the world. In this study, we are interested in the histidine acid phosphatase (HAP) family phytases present in insect-cultivated fungus gardens. Using bioinformatic approaches, 11 putative HAP phytase genes were initially screened from 18 publicly available metagenomes of fungus gardens and were further overexpressed in Escherichia coli. One phytase from a south pine beetle fungus garden showed the highest activity and was then chosen for further study. Biochemical characterization showed that the phytase is mesophilic but possesses strong ability to withstand high temperatures. To our knowledge, it has the longest half-life time at 100 °C (27 min) and at 80 °C (2.1 h) as compared to all the thermostable phytases publicly reported to date. After 100 °C incubation for 15 min, more than 93 % of the activity was retained. The activity was 3102 μmol P/min/mg at 37 °C and 4135 μmol P/min/mg at 52.5 °C, which is higher than all the known thermostable phytases. For the high activity level demonstrated at mesophilic temperatures as well as the high resilience to high temperatures, the phytase might be promising for potential application as an additive enzyme in animal feed.
KeywordsPhytase Metagenomic Overexpression Heat-resilient Mesophilic
This research was supported by the following grants: the Special Fund for Strategic Emerging Industry Development of Sichuan Province (SC2013510104007) and the SAAS Young Scientists Fund (2015QNJJ-015).
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors. This is the original work of the authors. The work described has not been submitted elsewhere for publication, in whole or in part. All authors confirm that ethical principles have been followed in the research as well as in manuscript preparation, and approved this submission.
Conflict of interest
All the authors (Hao Tan, Xiang Wu, Liyuan Xie, Zhongqian Huang, Weihong Peng, Bingcheng Gan) declare that they have no conflict of interest.
- Adedokun SA, Owusu-Asiedu A, Ragland D, Plumstead P, Adeola O (2015) The efficacy of a new 6-phytase obtained from Buttiauxella spp. expressed in Trichoderma reesei on digestibility of amino acids, energy, and nutrients in pigs fed a diet based on corn, soybean meal, wheat middlings, and corn distillers’ dried grains with solubles. J Anim Sci 93:168–175. doi: 10.2527/jas.2014-7912 CrossRefPubMedGoogle Scholar
- Ariza A, Moroz OV, Blagova EV, Turkenburg JP, Waterman J, Roberts SM, Vind J, Sjøholm C, Lassen SF, De Maria L, Glitsoe V, Skov LK, Wilson KS (2013) Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study. PLoS One 8:e65062. doi: 10.1371/journal.pone.0065062 PubMedCentralCrossRefPubMedGoogle Scholar
- 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. doi: 10.1111/j.1742-4658.2010.07559.x CrossRefPubMedGoogle Scholar
- Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258. doi: 10.1093/nar/gku340 PubMedCentralCrossRefPubMedGoogle Scholar
- Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, Hazlewood GP, Short JM, Robertson DE, Gray KA (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. doi: 10.1128/aem.70.5.3041-3046.2004 PubMedCentralCrossRefPubMedGoogle Scholar
- 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 23:1017–1021Google Scholar
- 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. doi: 10.1007/s12010-014-1440-y CrossRefPubMedGoogle Scholar
- Huang H, Shi P, Wang Y, Luo H, Shao N, Wang G, Yang P, Yao B (2009b) Diversity of beta-propeller phytase genes in the intestinal contents of grass carp provides insight into the release of major phosphorus from phytate in nature. Appl Environ Microbiol 75:1508–1516. doi: 10.1128/AEM.02188-08 PubMedCentralCrossRefPubMedGoogle Scholar
- Huang H, Zhang R, Fu D, Luo J, Li Z, Luo H, Shi P, Yang P, Diao Q, Yao B (2011) Diversity, abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation. Environ Microbiol 13:747–757. doi: 10.1111/j.1462-2920.2010.02379.x CrossRefPubMedGoogle Scholar
- Inoue H, Fujii T, Yoshimi M, Taylor LE II, Decker SR, Kishishita S, Nakabayashi M, Ishikawa K (2013) Construction of a starch-inducible homologous expression system to produce cellulolytic enzymes from Acremonium cellulolyticus. J Ind Microbiol Biotechnol 40:823–830. doi: 10.1007/s10295-013-1286-2 CrossRefPubMedGoogle Scholar
- Markowitz VM, Ivanova NN, Szeto E, Palaniappan K, Chu K, Dalevi D, Chen IMA, Grechkin Y, Dubchak I, Anderson I, Lykidis A, Mavromatis K, Hugenholtz P, Kyrpides NC (2008) IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res 36:D534–D538. doi: 10.1093/nar/gkm869 PubMedCentralCrossRefPubMedGoogle Scholar
- 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. doi: 10.1016/j.ejps.2010.09.019 CrossRefPubMedGoogle Scholar
- Nam S-J, Kim Y-O, Ko T-K, Kang J-K, Chun K-H, Auh J-H, Lee I-K, Park S, Oh BC (2014) Molecular and biochemical characteristics of beta-propeller phytase from marine Pseudomonas sp. BS10-3 and its potential application for animal feed additives. J Microbiol Biotechnol 24:1413–1420. doi: 10.4014/jmb.1407.07063 CrossRefPubMedGoogle Scholar
- Nedeva R, Knev M (1993) A research of the effect of the combined feeds with different participation of calcium and phosphorus for growing pigs. Zhivotnov"dni Nauk 30:61–66Google Scholar
- Tan H, Barret M, Mooij MJ, Rice O, Morrissey JP, Dobson AD, Griffiths BS, O’Gara F (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. doi: 10.1007/s00374-012-0755-5 CrossRefGoogle Scholar
- 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. doi: 10.1016/j.biortech.2010.01.001 CrossRefPubMedGoogle Scholar
- Zhang R, Yang P, Huang H, Yuan T, Shi P, Meng K, Yao B (2011) Molecular and biochemical characterization of a new alkaline β-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115. Appl Microbiol Biotechnol 92:317–325. doi: 10.1007/s00253-011-3309-0 CrossRefPubMedGoogle Scholar
- 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. doi: 10.1128/aem.02970-06 PubMedCentralCrossRefPubMedGoogle Scholar