Bacterial chitinases has a major role in chitinaceous waste management, biological control of pests and phytopathogens. In the present study, exochitinase gene ChitA encoding extracellular chitinase from the mangrove bacteria Bacillus pumilus MCB-07 was genetically characterized. Oligonucleotide primers specific to chitinase gene of Bacillus pumilus were designed and amplified by PCR. The purified PCR product was successfully cloned in pGEM-T vector and transformed into Escherichia coli DH5-α competent cells. Nucleotide sequence alignment of the chitinase gene revealed 96 % similarity whereas 94 % of the catalytic domain of 598 amino acids is conserved with protein family GH18 chitinases, which is a novel report for Bacillus pumilus. The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which demonstrated that chitinase of Bacillus pumilus MCB-07 is a novel gene. Multiple sequence alignment of chitinase gene sequences and its predicted amino acid sequences were also evaluated and the sequence was deposited in GenBank with accession number KT966736.1. Homology modeling of the chitinase depicted the typical (α/β) 8 TIM barrel structure. Molecular docking of the protein was performed by Autodock 4.2.6 and the docked pocket contained Val 113, Met 114, Gln 99, Ala 75 and Cys 98 as the key binding residues. The molecular docking of Bacillus pumilus chitinase, revealed the involvement of a phenylalanine of the catalytic domain in the catalytic process of chitin to mono and oligomers of NAG. The amino acid exhibited both hydrophobic and hydrogen bond interactions of chitin molecules with phenylalanine.

中文翻译：

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新型红树林分离株短小芽孢杆菌MCB-7胞外几丁质酶的序列分析和对接性能

细菌几丁质酶在几丁质废物管理、害虫和植物病原体的生物防治中具有重要作用。在本研究中，编码来自红树林细菌短小芽孢杆菌 MCB-07 的胞外几丁质酶的外切几丁质酶基因 ChitA 被遗传表征。设计特异性针对短小芽孢杆菌几丁质酶基因的寡核苷酸引物并通过PCR扩增。纯化后的 PCR 产物成功克隆到 pGEM-T 载体中，并转化到大肠杆菌 DH5-α 感受态细胞中。几丁质酶基因的核苷酸序列比对显示 96% 的相似性，而 598 个氨基酸的催化结构域的 94% 与蛋白质家族 GH18 几丁质酶保守，这是短小芽孢杆菌的新报告。插入物还显示出与其他 sp 的许多替换（突变）。芽孢杆菌的结果表明短小芽孢杆菌 MCB-07 的几丁质酶是一个新基因。还评估了几丁质酶基因序列及其预测氨基酸序列的多序列比对，并将该序列保存在GenBank中，登录号为KT966736.1。几丁质酶的同源建模描绘了典型的 (α/β) 8 TIM 桶状结构。蛋白质的分子对接通过 Autodock 4.2.6 进行，对接的口袋包含 Val 113、Met 114、Gln 99、Ala 75 和 Cys 98 作为关键结合残基。短小芽孢杆菌几丁质酶的分子对接揭示了催化结构域的苯丙氨酸参与几丁质到 NAG 的单聚体和寡聚体的催化过程。氨基酸表现出几丁质分子与苯丙氨酸的疏水和氢键相互作用。