为提高米曲霉(Aspergillus oryzae)糖苷水解酶家族(GHF) 11木聚糖酶AoXyn11A的耐热性,将其N-端置换为来源于嗜热裂孢菌(Thermobifida fusca)的同一家族耐热木聚糖酶pXYL11的对应区域.基于木聚糖酶耐热性的理性设计,采用大引物PCR技术将AoXyn 11A基因(Aoxyn11A)的5'-端DNA片段置换为pXYL11人工合成基因(xyn11PM)的对应片段,构建出杂合木聚糖酶ATX11A基因(ATx11A).分别将Aoxyn11A和ATx11A在毕赤酵母GS115中进行了表达,并分析了重组表达产物AoXyn 11A和ATX11A的温度特性.结果表明:ATX11A的最适温度Topt由AoXyn11A的50℃提升至65℃,在60℃的半衰期t1/260为55 rain,较AoXyn11A延长了41.3倍;ATX11A在55℃处理3h保留60％以上的酶活性,而AoXyn11A处理15 min酶活性完全丧失.本研究通过N-端置换显著改善了AoXyn11A的温度特性.%To enhance the thermotolerance of AoXyn 11 A,a glycoside hydrolase family (GHF) 11 mesophilic xylanase from Aspergillus oryzae,its N-terminal region was substituted with the corresponding one of the same family thermostable xylanase pXYL11 from Thermobifida fusca NTU22.Based on the rational design of xylanase thermotolerance,an ATX11A-encoding gene (A Tx1 1A) was constructed by replacing the 5'-end DNA fragment of AoXyn 11A gene (A oxyn1 1A) with the corresponding one of the synthesized pXYL11 gene (xyn11PM) using the megaprimer PCR technique.Aoxyn11A and A Tx11A were expressed in Pichia pastoris GS115,respectively,and the temperature characteristics of the expressed recombinant products,AoXyn1 1A and ATX11A,were analyzed.The results indicated that the temperature optimum (Topt) of hybrid xylanase ATX 11A was 65 ℃,which was 15 ℃ higher than that ofAoXynl 1A.Its half-life at 60 ℃ (t1/260) was 55 min,which was 41.3-fold longer than that of AoXyn 11A.When incubated at 55 ℃,ATX 11A retained more than 60％ of its original activity for 3 h,while AoXynl 1A entirely lost its activity only for 15 min.In this work,the temperature characteristics of AoXyn1 1A were significantly improved by N-terminus replacement.
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