期刊
ENZYME AND MICROBIAL TECHNOLOGY
卷 140, 期 -, 页码 -出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2020.109624
关键词
Chitinase; Cloning; Homology modeling; Bacillus pumilus; Molecular docking
资金
- UniBiosys Biotech Research Labs
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 colt DH5-alpha 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 (alpha/beta) 8 TIM barrel structure. Molecular docking of the protein was performed by Autodock 4.2.6 and the docked pocket contained Val 113, Ma 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.
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