定点饱和突变改善米曲霉木聚糖酶的温度特性  

Improvement in the Temperature Properties of Xylanase by Site-Saturated Mutagenesis

作　　者：吴芹[1] 臧嘉[1] 胡蝶[1] 王瑞[1] 邬敏辰 WU Qin;ZANG Jia;HU Die;WANG Rui;WU Minchen(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Wuxi Medical School,Jiangnan University,Wuxi 214122,China)

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：《食品与生物技术学报》2019年第7期65-70,共6页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目（31271811）

摘　　要：为改善米曲霉木聚糖酶AoXyn11A的温度特性,基于其三维结构的同源建模和分子动力学模拟,随机替换最高B-factor值位点处的Gly21。以重组表达质粒pET-28a-Aoxyn11A为模板,采用全质粒两步PCR技术对AoXyn11A基因(Aoxyn11A)中编码Gly21的密码子实施饱和突变,将pET-28a-Aoxyn11A各突变体转化E.coli BL21(DE3),构建了突变转化子文库。以酶的热稳定性为指标,从文库中筛选出最优突变转化子(E.coli/Aoxyn11AG21I)。DNA测序结果显示,E.coli/Aoxyn11AG21I表达一种由Ile21替换了Gly21的突变酶AoXyn11AG21I。温度特性分析表明:突变酶的最适温度由突变前的55℃提高至65℃;AoXyn11AG21I在55℃及以下稳定,较AoXyn11A提高了7℃。另外,AoXyn11A突变前后的pH特性改变不大。To improve the temperature properties of AoXyn11A,a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae CICC40186,the amino acid residue of Gly21 that possessing the highest B-factor value was replaced randomly based on the homology modeling of AoXyn11A and molecular dynamics(MD)simulation for its three-dimensional structure.Using the recombinant plasmid pET-28a-Aoxyn11A as a template,the codon of Gly21 in the xylanase-encoding gene(Aoxyn11A)was site-saturated mutagenesis with the two-stage whole-plasmid PCR technique.And then,the mutational transformant library was constructed by transforming the mutants of pET-28a-Aoxyn11A into E.coli BL21.In reference to the thermostability of enzymes,optimal mutational transformant(E.coli/Aoxyn11AG21I)was screened from library.The DNA sequencing results showed that E.coli/Aoxyn11AG21I expressed a mutant enzyme(AoXyn11AG21I)with the amino acid residue of Gly21 changed by Ile21.The analytical results indicated that the optimal temperature(Topt)of AoXyn11AG21I was 65℃,which was 10℃higher than that of AoXyn11A.AoXyn11AG21I was thermostable at or below 55℃,being 7℃higher than that of AoXyn11A.In addition,the pH properties of AoXyn11AG21I did not obviously change compared with AoXyn11A.

关 键 词：定点饱和突变 木聚糖酶 温度特性 分子动力学模拟 B-factor值 

分 类 号：Q936[生物学—微生物学]