引入盐桥改善木聚糖酶AoXyn11A的温度特性  

Improvement in the temperature properties of xylanase AoXyn11A by introducing three pairs of salt bridges

作　　者：吴芹[1] 臧嘉[1] 李闯[1] 王瑞[1] 邬敏辰[2] 

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122

出　　处：《食品与发酵工业》2016年第7期69-73,共5页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31271811)

摘　　要：为改善米曲霉(Aspergillus oryzae CICC40186)木聚糖酶Ao Xyn11A的温度特性,基于其与同一糖苷水解酶家族耐热木聚糖酶Ev Xyn11TS一级和三维结构的比对和分析,在Ao Xyn11A N端空间区域引入3对盐桥。采用大引物PCR技术对野生型木聚糖酶基因(Aoxyn11A)实施定点突变,构建突变酶Ao Xyn11AS基因(Aoxyn11AS)。分别将Aoxyn11A和Aoxyn11AS在毕赤酵母(Pichia pastoris)GS115中进行表达。酶学性质分析显示:Ao Xyn11AS的最适温度(Topt)为58℃,较Ao Xyn11A提高了8℃;突变酶在50℃的半衰期(t501/2)为60 min,较原酶延长了1倍多,其在55和60℃完全丧失酶活性的时间分别由原酶的15和4 min延长至35和15 min;而突变酶的p H特性和动力学常数较原酶改变不大。在Ao Xyn11A N端空间区域引入盐桥增加了其三维结构的刚性,从而明显改善了其温度特性。To improve the temperature properties of AoXyn 11 A, a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae CICC40186, three pairs of salt bridges were introduced into its N-terminal region based on the primary and three-dimensional （3-D） structure alignment and analysis between AoXyn 11A and the same family thermostable xylanase EvXynllTs. A mutant xylanase-encoding gene （AoxynllAs） was constructed through site-directed mutagenesis on wild-type xylanase-encoding gene （Aoxyn 11A） using the megaprimer PCR technique. Two genes, Aoxyn11A and Aoxyn11As, were respectively expressed in Pichia pastoris GS115. The analytical results of en- zymatic properties showed that the optimal temperature （ Topt） of AoXyn 11As was 58 ℃ , which was 8 ℃ higher than that of AoXyn11A. Its half-life at 50 ℃ （t 1/2 50） was 60 min, which was more than one-fold longer than that of AoXyn11A. When incubated at 55 and 60 ℃ , the periods that AoXyn11As entirely lost its activity extended from 15 and 4 min to 35 and 15 min, respectively. However, the pH properties and kinetic parameters of AoXyn 11As did not obviously change compared with AoXyn 11A. Introducing salt bridges into the N-terminal space region of AoXyn 11A could increase the rigidity of its 3-D structure, so that obviously improved its temperature properties.

关 键 词：盐桥 木聚糖酶 温度特性 结构分析 N端空间区域 定点突变 

分 类 号：TQ925[轻工技术与工程—发酵工程]