饱和突变改善米曲霉11家族木聚糖酶AEx11A的催化特性

doi:10.3969/j.issn.1673-1689.2020.05.005

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饱和突变改善米曲霉11家族木聚糖酶AEx11A的催化特性
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                        10.3969/j.issn.1673-1689.2020.05.005
                    
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Improvement in the Catalytic Properties of A GH 11 Xylanase AEx11A，from Aspergillus oryzae by Saturated Mutagenesis

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摘要:

为改善米曲霉（Aspergillus oryzae） 11家族木聚糖酶AEx11A的催化特性，作者采用全质粒PCR方法对AEx11A基因（AEx11A）中编码Thr98、Asn100、Val¹²⁴、Pro¹²⁹和Ile¹³²的密码子实施饱和突变，构建饱和突变文库。以木聚糖酶的酶活性为指标，采用DNS法从文库中筛选出酶活性提高30%以上的转化子。对其中酶活性提高最明显的两个位点Thr⁹⁸和Val¹²⁴实施迭代饱和突变，筛选出最优突变子AEx11^{AT98D-V124Q，}其纯化后的比活性及催化效率分别为AEx11A的3.04和2.74倍。此外，突变酶AEx11AV^124T的热稳定性较AEx11A有一定的提高，其余2个突变酶的温度特性与AEx11A差异不大。

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In order to improve the catalytic properties of AEx11A，a glycoside hydrolase（GH） family 11 xylanase from Aspergillus oryzae，the Thr⁹⁸，Asn¹⁰⁰，Val¹²⁴，Pro¹²⁹ and Ile¹³²-encoding codons in AEx11A were selected for saturated mutagenesis by whole-plasmid PCR technique. Then，the mutagenesis libraries of AEx11A were constructed by transforming these variants into E. coli BL21（DE3），respectively. Using the enzyme activity of xylanase as criterion，transformants having an increase of more than 30% in activities were selected from the libraries by DNS method. Two mutation sites（Thr⁹⁸ and Val¹²⁴） with remarkably increased enzyme activities were subjected to iterative saturation mutation（ISM） and the optimal mutant，AEx11^AT98D-V124Q，was selected. The specific activity and catalytic efficiency of purified AEx11^AT98D-V124Q were 3.04- and 2.74-fold those of AEx11A. An improvement in the thermostability of AEx11^AV124T was observed compared with that of AEx11A，while the temperature properties of the other two mutants almost had no changes.

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刘艳,张婷,阚婷婷,李剑芳,邬敏辰.饱和突变改善米曲霉11家族木聚糖酶AEx11A的催化特性[J].食品与生物技术学报,2020,39(5):31-37.

LIU Yan, ZHANG Ting, KAN Tingting, LI Jianfang, WU Minchen. Improvement in the Catalytic Properties of A GH 11 Xylanase AEx11A，from Aspergillus oryzae by Saturated Mutagenesis[J]. Journal of Food Science and Biotechnology,2020,39(5):31-37.

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在线发布日期: 2020-06-19
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