Increase in the thermostability of Bacillus sp strain TAR-1 xylanase using a site saturation mutagenesis library
Increase in the thermostability of Bacillus sp strain TAR-1 xylanase using a site saturation mutagenesis library
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DOI：
10.1080/09168451.2018.1495550
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发表时间：
2018-01-01
影响因子：
1.6
通讯作者：
Yasukawa, Kiyoshi
中科院分区：
文献类型：
--
作者：
Nakatani, Kota;Katano, Yuta;Yasukawa, Kiyoshi
位点饱和诱变文库是最近发展起来的一种技术，其中目标区域中所有氨基酸残基中的任何一个被替换为其他19个氨基酸残基。在这项研究中，我们使用这种技术来提高来自芽孢杆菌属的 GH10 木聚糖酶 XynR 的热稳定性。菌株TAR-1。我们假设XynR的底物结合区域是柔性的，并且如果在不损害底物结合能力的情况下降低底物结合区域的柔性，则XynR的热稳定性将会增加。构建了XynR的氨基酸残基Tyr43-Lys115和Ala300-Asn325的位点饱和诱变文库。通过筛选 480 个克隆，选择 S92E 作为最耐热的克隆，在雷马素亮蓝木聚糖的水解中，80℃热处理 15 分钟后，表现出 80% 的残留活性。我们的结果表明该策略对于稳定 GH10 木聚糖酶是有效的。缩写：DNS：3,5-二硝基水杨酸； RBB-木聚糖：雷马素亮蓝-木聚糖
Site saturation mutagenesis library is a recently developed technique, in which any one out of all amino acid residues in a target region is substituted into other 19 amino acid residues. In this study, we used this technique to increase the thermostability of a GH10 xylanase, XynR, from Bacillus sp. strain TAR-1. We hypothesized that the substrate binding region of XynR is flexible, and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Site saturation mutagenesis libraries of amino acid residues Tyr43-Lys115 and Ala300-Asn325 of XynR were constructed. By screening 480 clones, S92E was selected as the most thermostable one, exhibiting the residual activity of 80% after heat treatment at 80 degrees C for 15min in the hydrolysis of Remazol Brilliant Blue-xylan. Our results suggest that this strategy is effective for stabilization of GH10 xylanase.Abbreviations: DNS: 3,5-dinitrosalicylic acid; RBB-xylan: Remazol Brilliant Blue-xylan