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Die Hu,Jianfang Li,Qin Wu,Jia Zang,Jianqing Cheng,Minchen Wu 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.6
To improve the temperature characteristics of a mesophilic glycoside hydrolase family (GHF) 11 xylanase AoXyn11A from Aspergillus oryzae, both introduction of a disulfide bridge and the substitution of a specific amino acid were carried out by in silico design and site-directed mutagenesis. Based on the analysis of a known crystal structure of thermophilic xylanase TlXynA from Thermomyces lanuginosus, and the alignment of primary structures between AoXyn11A and TlXynA, one mutant AoXyn11AM with a disulfide bridge (Cys108–Cys152) was designed by replacing the Ser108 and Asn152 of AoXyn11A with Cys residues, respectively. Additionally, based on the analysis of amino acid B-factor values, another mutant AoXyn11AM-G22A was predicted by substituting Gly22 of AoXyn11AM (having the maximum B-factor value of 69.25 Å, with the corresponding Ala23 of TlXynA. Thereafter, two mutant xylanase-encoding genes, Aoxyn11AM and Aoxyn11AM-G22A, were constructed by site-directed mutagenesis. Aoxyn11A and two mutant genes were expressed in E. coli BL21(DE3) respectively, and three expressed recombinant xylanases, reAoXyn11A, reAoXyn11AM and reAoXyn11AM-G22A, were purified to homogeneity. The temperature optima of reAoXyn11AM and reAoXyn11AM-G22A were 60 and 65°C, respectively, being 5 and 10°C higher than that of reAoXyn11A. Their thermal inactivation half-lives at 50°C were 1.8- and 8.4-folds longer than that of reAoXyn11A. There were no obvious alterations after mutations in specific activity and enzymatic properties, except for the temperature characteristics.
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