Improving Hydrolysis Characteristics of Xylanases by Site-Directed Mutagenesis in Binding-Site Subsites from Streptomyces L10608.
Improving Hydrolysis Characteristics of Xylanases by Site-Directed Mutagenesis in Binding-Site Subsites from Streptomyces L10608.
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通过链霉菌 L10608 结合位点亚位点定点诱变改善木聚糖酶的水解特性
DOI：
10.3390/ijms19030834
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发表时间：
2018-03-13
影响因子：
5.6
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Xiong K;Xiong S;Gao S;Li Q;Sun B;Li X
木聚糖水解制备低聚糖是农业废弃物半纤维素原料增值的有效途径。从土壤中分离的细菌菌株链霉菌L10608含有编码葡糖苷水解酶家族10/11（GH 10/11）的木聚糖酶的基因，并且这些基因已被克隆以催化低聚木糖（XOS）的生产。为了提高木聚糖酶水解产物中XOS的比例，采用定点突变的方法对木聚糖酶的4个氨基酸残基进行了突变，并在大肠杆菌中进行了过量表达。突变将编码Asn 214（+2）和Asn 86（-2）的密码子替换为Ala，并去除了GH 10-xyn中的蓖麻毒素B-凝集素结构域，并产生了GH 11-xyn的突变体Y115 A（-2）和Y123 A（-2）。有趣的是，GH 10-N86 Q显著增加了XOS的水解，几乎消除了木糖（X1）至<2.5%，这表明L10608的GH 10-xyn的−2结合位点是与木三糖（X3）结合所必需的。使用山毛榉木聚糖作为底物，GH 10-N86 Q的水解活性增加约1.25倍，并且对底物具有高亲和力，Km约为1.85 mg·mL-1。除此之外，GH 10-N86 Q和GH 10-xyn之间的酶性质没有显着差异。这些突变体提供了巨大的潜力，改造木聚糖酶与所需的XOS水解。
The preparation of oligosaccharides via xylan hydrolysis is an effective way to add value to hemicellulosic material of agricultural waste. The bacterial strain Streptomyces L10608, isolated from soil, contains genes encoding xylanases of glucoside hydrolase family 10/11 (GH10/11), and these have been cloned to catalyze the production of xylooligosaccharide (XOS). To improve the XOS proportion of hydrolysates produced by xylanase, four amino acid residues were substituted by site-directed mutagenesis, and the mutant genes were overexpressed in Escherichia coli. Mutations replaced the codons encoding Asn214 (+2) and Asn86 (−2) by Ala and removed the Ricin B-lectin domain in GH10-xyn, and mutants Y115A (−2) and Y123A (−2) were produced for GH11-xyn. Interestingly, GH10-N86Q had significantly increased hydrolysis of XOS and almost eliminated xylose (X1) to <2.5%, indicating that the −2 binding site of GH10-xyn of L10608 is required for binding with xylotriose (X3). The hydrolytic activity of GH10-N86Q was increased approximately 1.25-fold using beechwood xylan as a substrate and had high affinity for the substrate with a low Km of about 1.85 mg·mL−1. Otherwise, there were no significant differences in enzymatic properties between GH10-N86Q and GH10-xyn. These mutants offer great potential for modification of xylanase with desired XOS hydrolysis.
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影响因子：
4.1
作者：
Boraston, AB;Tomme, P;Kilburn, DG
通讯作者：
Kilburn, DG
影响因子：
2.4
作者：
Paes, Gabriel;Tran, Vinh;O'Donohue, Michael J.
通讯作者：
O'Donohue, Michael J.
影响因子：
4.1
作者：
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通讯作者：
Kluepfel, D
影响因子：
4.1
作者：
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通讯作者：
OKADA, H
影响因子：
2.7
作者：
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通讯作者：
Chen, Yuanling