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

DOI：10.3969/j.issn.1673-1689.2019.07.009

中文关键词: 定点饱和突变 木聚糖酶 温度特性 分子动力学模拟 B-factor值

英文关键词: site-saturated mutagenesis,xylanase,temperature properties,molecular dynamics simulation,B-factor values

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作者

单位

吴芹

江南大学 生物工程学院，江苏 无锡 214122

臧嘉

江南大学 生物工程学院，江苏 无锡 214122

胡蝶

江南大学 生物工程学院，江苏 无锡 214122

王瑞

江南大学 生物工程学院，江苏 无锡 214122

邬敏辰

江南大学 无锡医学院，江苏 无锡 214122

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中文摘要:

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. coliBL21（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。

英文摘要:

To improve the temperature properties of AoXyn11A，a glycoside hydrolase family 11 mesophilic xylanase fromAspergillusoryzae CICC40186，the amino acid residue of Gly21that 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 Gly21in 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 intoE. coliBL21. In reference to the thermostability of enzymes，optimal mutational transformant（E. coli/Aoxyn11AG21I） was screened from library. The DNA sequencing results showed thatE. coli/Aoxyn11AG21Iexpressed a mutant enzyme（AoXyn11AG21I） with the amino acid residue of Gly21changed by Ile21. The analytical results indicated that the optimal temperature（Topt） of AoXyn11AG21Iwas 65 ℃，which was 10 ℃ higher than that of AoXyn11A. AoXyn11AG21Iwas 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.

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