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
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