定点饱和突变改善米曲霉木聚糖酶的温度特性

doi:10.3969/j.issn.1673-1689.2019.07.009

首页 > 过刊浏览>2019年第38卷第7期 >65-70. DOI:10.3969/j.issn.1673-1689.2019.07.009

定点饱和突变改善米曲霉木聚糖酶的温度特性
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                        10.3969/j.issn.1673-1689.2019.07.009
                    
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Improvement in the Temperature Properties of Xylanase by Site-Saturated Mutagenesis

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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/Aoxyn11A^G21I）。DNA测序结果显示，E. coli/Aoxyn11A^G21I表达一种由Ile²¹替换了Gly²¹的突变酶AoXyn11A^G21I。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。

Abstract:

To improve the temperature properties of AoXyn11A，a glycoside hydrolase family 11 mesophilic xylanase from Aspergillus oryzae 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 into E. coli BL21. In reference to the thermostability of enzymes，optimal mutational transformant（E. coli/Aoxyn11A^G21I） was screened from library. The DNA sequencing results showed that E. coli/Aoxyn11A^G21I expressed a mutant enzyme（AoXyn11A^G21I） with the amino acid residue of Gly²¹ changed by Ile²¹. The analytical results indicated that the optimal temperature（T^opt） of AoXyn11A^G21Iwas 65 ℃，which was 10 ℃ higher than that of AoXyn11A. AoXyn11A^G21Iwas 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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吴芹,臧嘉,胡蝶,王瑞,邬敏辰.定点饱和突变改善米曲霉木聚糖酶的温度特性[J].食品与生物技术学报,2019,38(7):65-70.

WU Qin, ZANG Jia, HU Die, WANG Rui, WU Minchen. Improvement in the Temperature Properties of Xylanase by Site-Saturated Mutagenesis[J]. Journal of Food Science and Biotechnology,2019,38(7):65-70.

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