N-端置换提高木聚糖酶AoXyn11A的耐热性

Enhancement in the Thermotolerance of Xylanase(AoXyn11A)by N-Terminus Replacement

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AoXyn11A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AoXyn11A和ATX11A的温度特性。结果表明:ATX11A的最适温度T_(opt)由AoXyn11A的50℃提升至65℃,在60℃的半衰期t_(1/2)^(60)为55 min,较AoXyn11A延长了41.3倍;ATX11A在55℃处理3 h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 AoXyn11A,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(ATx11A) was constructed by replacing the 5′-end DNA fragment of AoXyn11A gene(Aoxyn11A)with the corresponding one of the synthesized pXYL11 gene(xyn11PM) using the megaprimer PCR technique.Aoxyn11A and ATx11A were expressed in Pichia pastoris GS115,respectively,and the temperature characteristics of the expressed recombinant products,AoXyn11A and ATX11A,were analyzed.The results indicated that the temperature optimum(T_(opt)) of hybrid xylanase ATX11A was 65 ℃,which was 15 ℃ higher than that of AoXyn11A.Its half-life at 60 ℃(t_(1/2)^(60)) was 55 min,which was 41.3-fold longer than that of AoXyn11A.When incubated at 55 ℃,ATX11A retained more than 60% of its original activity for 3 h,while AoXyn11A entirely lost its activity only for 15 min.In this work,the temperature characteristics of AoXyn11A were significantly improved by N-terminus replacement.