Y13F定点突变改良米曲霉中温木聚糖酶的耐热性

Site-directed Mutagenesis of Y13F to Improve the Thermotolerance of Mesophilic Xylanase from Aspergillus oryzae

为改良米曲霉(Aspergillus oryzae)糖苷水解酶11家族木聚糖酶AoXyn11A的耐热性,将其Tyr^(13)(Y13)置换为Phe(F)。基于AoXyn11A与同一家族7种耐热木聚糖酶一级结构的多序列同源比对及其三维结构的同源建模和分子动力学模拟,设计了一种突变酶AoXyn11A^(Y13F);以重组质粒p PIC9K-Aoxyn11A为模板,采用PCR技术将AoXyn11A基因(Aoxyn11A)中编码Y13的密码子TAC突变为F的TTC,构建了一种突变酶基因(Aoxyn11A^(Y13F));分别将Aoxyn11A和Aoxyn11A^(Y13F)在毕赤酵母(Pichia pastoris)GS115中实施了表达,并对重组表达产物AoXyn11A和AoXyn11A^(Y13F)的耐热性进行了分析。结果表明:突变酶的最适温度T_(opt)由突变前的50℃提高到55℃;AoXyn11A^(Y13F)在50℃的半衰期t_(1/2)^(50)为95 min,较AoXyn11A(t_(1/2)^(50)=6 min)延长了约15倍。由此经Y13F定点突变显著改良了野生型木聚糖酶的耐热性。

To improve the thermotolerance of AoXyn11A,a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae,the amino acid residue Tyr^13（ 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 AoXyn11A^Y13F was designed. Using p PIC9K-Aoxyn11 A as a template,a mutant xylanase-encoding gene Aoxyn11A^Y13F was constructed by mutating a Y13-encoding codon TAC of Aoxyn11 A into a F-encoding TTC with PCR technique. Then,Aoxyn11 A and Aoxyn11A^Y13Fwere expressed in Pichia pastoris GS115,respectively,and the thermotolerance of expressed recombinant products,AoXyn11A and AoXyn11A^Y13F,were analyzed. The results indicated that the temperature optimum（ Topt） of AoXyn11A^Y13Fwas 55℃,higher than that（ 50℃） of AoXyn11A,and that the half-life at 50℃（ t1/2^50） was 95 min,which was 15 fold longer than that（ t1/2^50= 6min） of AoXyn11A. The thermotolerance of wild-type AoXyn11A were significantly improved by site-directed mutagenesis of Y13 F.