Wu Mei Li, Tsai Chun Yung, Chen Tsai Hsia
Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan.
FEMS Microbiol Lett. 2006 Mar;256(1):119-25. doi: 10.1111/j.1574-6968.2006.00115.x.
The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.
嗜水气单胞菌JP10中编码几丁质酶92（Chi92）的基因已利用冰核蛋白（INPN）的N端区域作为锚定基序展示在大肠杆菌的细胞表面。免疫荧光显微镜检查证实Chi92锚定在细胞表面。蛋白质免疫印迹分析进一步鉴定了预期大小（112 kDa）的包含N端结构域INPN-Chi92融合蛋白的INP衍生物的合成。全细胞酶活性测定表明展示的Chi92对胶体几丁质具有增强的催化活性。此外，展示Chi92的细胞对包括十细胞镰刀菌、茄丝核菌、立枯丝核菌和尖孢镰刀菌黄瓜专化型在内的植物病原真菌的菌丝生长表现出抑制作用。这项研究表明，基于INP的展示系统可用于在大肠杆菌细胞表面表达大蛋白（90 kDa的Chi92）而不抑制生长。此外，在细胞表面展示几丁质酶可能为开发针对植物病原真菌的生物防治剂提供一种有吸引力的方法。
