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| Site-directed Mutagenesis of Y13F to Improve the Thermotolerance of Mesophilic Xylanase from Aspergillus oryzae |
| WU Qin1, HU Die1, LI Xue-qing2, YUAN Feng-jiao2, LI Jian-fang2, WU Min-chen3 |
1. School of Biotechnology, Jiangnan University, Wuxi 214122, China;
2. School of Food Science and Technology, Jiangnan University, Wuxi 214122, China;
3. Wuxi Medical School, Jiangnan University, Wuxi 214122, China |
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Abstract To improve the thermotolerance of AoXyn11A, a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae, the amino acid residue Tyr13(Y13) was replaced by Phe (F). Based on the multiple alignment of the primary structures of AoXyn11A and seven thermophilic xylanases, the homology modeling of AoXyn11A and molecular dynamics (MD) simulation on its three-dimensional structure, a mutant enzyme AoXyn11AY13F was designed. Using pPIC9K-Aoxyn11A as a template, a mutant xylanase-encoding gene Aoxyn11AY13F was constructed by mutating a Y13-encoding codon TAC of Aoxyn11A into a F-encoding TTC with PCR technique. Then, Aoxyn11A and Aoxyn11AY13F were expressed in Pichia pastoris GS115, respectively, and the thermotolerance of expressed recombinant products, AoXyn11A and AoXyn11AY13F, were analyzed. The results indicated that the temperature optimum (Topt) of AoXyn11AY13F was 55℃, higher than that (50℃) of AoXyn11A, and that the half-life at 50℃ (t1/250) was 95 min, which was 15 fold longer than that (t1/250=6 min) of AoXyn11A. The thermotolerance of wild-type AoXyn11A were significantly improved by site-directed mutagenesis of Y13F.
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Received: 06 June 2016
Published: 25 December 2016
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