Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities.
Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities.
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苏云金芽孢杆菌几丁质酶的分子对接和定点诱变，以提高几丁质分解、协同鳞翅目杀幼虫和杀线虫活性
DOI：
10.7150/ijbs.10632
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发表时间：
2015
影响因子：
9.2
通讯作者：
Li L
中科院分区：
文献类型：
--
作者：
Ni H;Zeng S;Qin X;Sun X;Zhang S;Zhao X;Yu Z;Li L
细菌几丁质酶可用于农业上重要的害虫和真菌病原体的生物防治。然而，天然存在的细菌几丁质酶的效用通常受到其低酶活性的限制。在这项研究中，我们构建了突变体的苏云金芽孢杆菌几丁质酶增强活性的基础上同源建模，分子对接，和定点突变的目标残基修改的空间位置，空间位阻，或亲水性/疏水性。我们首先从B中鉴定出一个基因。苏云金杆菌YBT-9602，其编码属于具有保守的底物结合和底物催化基序的糖基水解酶家族18的几丁质酶（Chi 9602）。以环状芽孢杆菌1 ITX蛋白为模板，构建了含有底物结合结构域的Chi 9602（Chi 960235 -459）的截短型结构模型。我们使用二-N-乙酰基-D-葡萄糖胺作为配体对Chi 960235 -459进行了分子对接分析。然后，我们选择了10个感兴趣的残基从对接区的定点诱变实验和大肠杆菌中的表达。几丁质酶活性测定结果表明，这三个突变体的几丁质酶活性均有所提高。与野生型Chi 9602相比，ChiW 50 A突变体表现出几丁质分解活性增加超过60%，具有相似的pH、温度和金属离子需求。此外，ChiW 50 A还具有一定的杀虫活性和抑菌活性。该突变体与B具有显著的协同效应。苏云金芽孢晶体制剂对棉铃虫和秀丽隐杆线虫幼虫有明显的杀虫活性，对几种植物病原真菌有明显的杀虫活性。
Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.
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影响因子：
2.9
作者：
Chavan, S. B.;Deshpande, M. V.
通讯作者：
Deshpande, M. V.
DOI：
10.1155/2012/135498
发表时间：
2012
期刊：
Biotechnology research international
影响因子：
--
作者：
Kuzu SB;Güvenmez HK;Denizci AA
通讯作者：
Denizci AA
DOI：
10.1016/j.vascn.2004.04.002
发表时间：
2004-11-01
影响因子：
1.9
作者：
Dengg, Marlene;van Meel, Jacques C. A.
通讯作者：
van Meel, Jacques C. A.
苏云金芽孢杆菌突变株 BMB171 的完整基因组序列
影响因子：
3.2
作者：
He, Jin;Shao, Xiaohu;Yu, Ziniu
通讯作者：
Yu, Ziniu
影响因子：
3.7
作者：
Li H;Greene LH
通讯作者：
Greene LH