Department of Molecular Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.
Enzyme Microb Technol. 2011 Mar 7;48(3):225-31. doi: 10.1016/j.enzmictec.2010.11.001. Epub 2010 Nov 12.
Bacillus pumilus SG2 isolated from high salinity ecosystem in Iran produces two chitinases (ChiS and ChiL) and secretes them into the medium. In this study, chiS and chiL genes were cloned in pQE-30 expression vector and were expressed in the cytoplasm of Escherichia coli strain M15. The recombinant proteins were purified using Ni-NTA column. The optimum pH and optimum temperature for enzyme activity of ChiS were pH 6, 50°C; those of ChiL were pH 6.5, 40°C. The purified chitinases showed antifungal activity against Fusarium graminearum, Rhizoctonia solani, Magnaporthe grisea, Sclerotinia sclerotiorum, Trichoderma reesei, Botrytis cinerea and Bipolaris sp. Moreover, purified ChiS was identified as chitinase/lysozyme, which are capable of degrading the chitin component of fungal cell walls and the peptidoglycan component of cell walls with many kinds of bacteria (Xanthomonas translucens pv. hordei, Xanthomonas axonopodis pv. citri, Bacillus licheniformis, E. coli C600, E. coli TOP10, Pseudomonas aeruginosa and Pseudomonas putida). Strong homology was found between the three-dimensional structures of ChiS and a chitinase/lysozyme from Bacillus circulans WL-12. This is the first report of a bifunctional chitinase/lysozyme from B. pumilus.
从伊朗高盐环境中分离出的短小芽孢杆菌 SG2 产生两种几丁质酶（ChiS 和 ChiL）并将其分泌到培养基中。在本研究中，chiS 和 chiL 基因被克隆到 pQE-30 表达载体中，并在大肠杆菌 M15 菌株的细胞质中表达。重组蛋白使用 Ni-NTA 柱进行纯化。ChiS 的最适 pH 和最适温度为 pH6、50°C；ChiL 的最适 pH 和最适温度为 pH6.5、40°C。纯化的几丁质酶对禾谷镰刀菌、茄病镰刀菌、稻瘟病菌、核盘菌、里氏木霉、灰葡萄孢和双极菌表现出抗真菌活性。此外，纯化的 ChiS 被鉴定为几丁质酶/溶菌酶，能够降解真菌细胞壁的几丁质成分和多种细菌细胞壁的肽聚糖成分（禾谷多黏菌、柑橘溃疡病菌、地衣芽孢杆菌、大肠杆菌 C600、大肠杆菌 TOP10、铜绿假单胞菌和恶臭假单胞菌）。ChiS 的三维结构与来自环状芽孢杆菌 WL-12 的几丁质酶/溶菌酶具有很强的同源性。这是短小芽孢杆菌来源的双功能几丁质酶/溶菌酶的首次报道。
