Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

AMB Express
Xueqing LiJian-Fang Li

Abstract

To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly(21) with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A(G21I), was selected from the mutagenesis library, E. coli/Aoxyn11A (G21X) (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11A(Y13F) or AoXyn11A(G21I-Y13F), was designed by replacing Tyr(13) in AoXyn11A or AoXyn11A(G21I) with Phe. Finally, three variant-encoding genes, Aoxyn11A (G21I), Aoxyn11A (Y13F) and Aoxyn11A (G21I-Y13F), were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T opt) of recombinant (re) AoXyn11A(G21I-Y13F) was 60 °C, being 5 °C higher than that of reAoXyn11A(G21I) or reAoXyn11A(Y13F), and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t 1/2...Continue Reading

References

Jan 23, 1999·Current Opinion in Structural Biology·R Jaenicke, G Böhm
Apr 21, 2007·Nature Protocols·Manfred T Reetz, José Daniel Carballeira
Jun 3, 2008·The Journal of Biological Chemistry·Claire DumonHarry J Gilbert
Jul 10, 2009·Protein Engineering, Design & Selection : PEDS·M S MadhusudhanAndrej Sali
Jul 31, 2009·Bioresource Technology·Bassam Al BalaaIsabelle Housen
Sep 11, 2010·Current Opinion in Chemical Biology·Jonathan K Lassila
Jan 29, 2011·Journal of Bioscience and Bioengineering·Thayat SriyapaiKosum Chansiri
Aug 3, 2011·Biotechnology and Bioengineering·Somayesadat BadieyanChenming Zhang
Mar 31, 2012·Journal of Industrial Microbiology & Biotechnology·Feng ZhangWen-Jun Li
Sep 4, 2012·Protein Engineering, Design & Selection : PEDS·H LiO Turunen
Sep 3, 2013·Applied Microbiology and Biotechnology·Inês R SilvaJørn D Mikkelsen
Aug 6, 2014·Journal of Biotechnology·Dawn Elizabeth StephensKugen Permaul
Aug 21, 2014·Applied Biochemistry and Biotechnology·Misumi KataokaKazuhiko Ishikawa
Jan 13, 2016·World Journal of Microbiology & Biotechnology·Vishal KumarPratyoosh Shukla
Jul 14, 2016·Biochemistry·Masahiro WatanabeKazuhiko Ishikawa

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Citations

Jan 19, 2018·Critical Reviews in Biotechnology·Abdul BasitHuiqiang Lou
Jul 26, 2018·Chembiochem : a European Journal of Chemical Biology·Aitao LiManfred T Reetz

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Methods Mentioned

BETA
PCR
electrophoresis
Protein Assay
thermal shift

Software Mentioned

PyMol
FITTER
SALIGN
MODELLER
CASTER
GROMACS
Quantity One
rms
ClustalW2
Origin

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