Abstract
Due to our previous research, mainly the thermostable mutants Q307D, Y311K, and I427L, we conjectured that Escherichia coli AppA phytase’s C-terminal plays an important role in its thermostability, and AppA begins to collapse from the C-terminal when at a higher temperature. So here we constructed C-lose mutant to prove it. The residual activities of the wild-type AppA phytase and C-lose were 31.42 and 70.49 %, respectively, after being heated at 80 °C for 10 min. The C-terminal deletion mutant C-lose showed 39.07 % thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved the C-lose plays a key role in E. coli AppA phytase’s thermostability.
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Eijsink VG, Bjørk A, Gåseidnes S, Sirevåg R, Synstad B, van den Burg B, Vriend G (2004) Rational engineering of enzyme stability. J Biotechnol 113:105–120
Garrett JB, Kretz KA, Donoghue EO, 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(5):3041–3046
Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O (2005) Biotechnological production and applications of phytases. Appl Microbiol Biotechnol 68(5):588–597
Han YM, Lei XG (1999) Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Arch Biochem Biophys 364:83–90
Han YM, Wilson DB, Lei XG (1999) Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Appl Environ Microbiol 65:1915–1918
Kim MS, Lei XG (2008) Enhancing thermostability of Escherichia coli phytase App A2 by error-prone PCR. Appl Microbiol Biotechnol 79(1):69–75
Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191
Lei XG, Stahl CH (2001) Biotechnological development of effective phytases for mineral nutrition and environmental protection. Appl Microbiol Biotechnol 57(4):474–481
Lim D, Golovan S, Forsberg CW, Jia Z (2000) Crystal structures of Escherichia coli phytase and its complex with phytate. Nat Struct Biol 7(2):108–113
Luo H, Huang H, Yang P, Wang Y, Yuan T, Wu N, Yao B, Fan Y (2007) A novel phytase appA from Citrobacter amalonaticus CGMCC 1696: gene cloning and overexpression in Pichia pastoris. Curr Microbiol 55(3):185–192
Pack SP, Yoo YJ (2003) Protein thermostability: structure-based difference of residual properties between thermophilic and mesophilic proteins. J Mol Catal B Enzym 26:257–264
Querol E, Perez-Pons JA, Mozo-Villarias A (1996) Analysis of protein conformational characteristics related to thermostability. Protein Eng 9:265–271
Ragone R (2001) Hydrogen-bonding classes in proteins and their contribution to the unfolding reaction. Protein Sci 10:2075–2082
Rodriguez E, Wood ZA, Karplus PA, Lei XG (2000) Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. Arch Biochem Biophys 382(1):105–112
Selle PH, Aaron J, Cowieson AJ, Ravindran V (2009) Consequences of calcium interactions with phytate and phytase for poultry and pigs. Livestock Sci 124(1–3):126–141
Szilágyi A, Závodszky P (2000) Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure 15:493–504
Vieille C, Zeikus JG (1996) Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol 14:183–190
Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43
Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, Lehmann M, van Loon AP (1999) Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 65(2):367–373
Yip KS, Stillman TJ, Britton KL, Artymiuk PJ, Baker PJ, Sedelnikova SE, Engel PC, Pasquo A, Chiaraluce R, Consalvi V (1995) The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures. Structure 3:1147–1158
Zhu W, Qiao D, Huang M, Yang G, Xu H, Cao Y (2010) Modifying thermostability of appA from Escherichia coli. Curr Microbiol 61(4):267–273
Acknowledgments
This study was supported by National Science Funds Committee (31272659, 30971817), and National twelfth five-year science and technology support program (2011BAD14B05), Sichuan Science and Technology Bureau (2010GZ0290, 2012GZ0008, 2010GZ0065, 2011GZ0027).
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Fei, B., Cao, Y., Xu, H. et al. AppA C-terminal Plays an Important Role in its Thermostability in Escherichia coli . Curr Microbiol 66, 374–378 (2013). https://doi.org/10.1007/s00284-012-0283-4
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DOI: https://doi.org/10.1007/s00284-012-0283-4