Effects of chitin binding domain on enzymatic properties and insecticidal activity of Bombyx mori chitinase
Effects of chitin binding domain on enzymatic properties and insecticidal activity of Bombyx mori chitinase
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几丁质结合域对家蚕几丁质酶酶学性质和杀虫活性的影响
DOI：
10.1007/s11274-010-0607-0
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发表时间：
2011-07-01
影响因子：
4.1
通讯作者：
Pei, Yan
中科院分区：
文献类型：
--
作者：
Fan, Yanhua;Guo, Shujun;Pei, Yan
家蚕几丁质酶（Bmchi）具有催化结构域和富含半胱氨酸的c端结构域。从重组毕赤酵母宿主中表达并纯化了野生型和c端截断型（Bmchia dagger c）。c端结构域的缺失降低了几丁质酶结合和降解不溶性底物的能力。研究还发现Bmchi和Bmchia dagger c在最佳pH和温度、热稳定性以及降解某些寡糖底物的能力方面存在差异。为了确定这些蛋白是否可以用于提高昆虫病原真菌的毒力，我们在A. nidulans gpdA组成启动子的控制下，将Bmchi和Bmchia dagger c引入球孢博维拉（Beauveira bassiana）中。利用WT和转化后的球孢白僵菌进行昆虫生物测定，结果显示，与WT亲本菌株相比，表达Bmchi显著提高了球孢白僵菌的毒力，而Bmchia dagger c在球孢白僵菌中的表达只有名义上的效果。这些结果表明，mori几丁质酶可以用来提高真菌的效率，但c端结构域对这一功能至关重要。
Bombyx mori chitinase (Bmchi) possesses a catalytic domain and a cysteine-rich C-terminal domain. Wild-type and a C-terminal truncated form (Bmchia dagger c) were expressed and purified from recombinant Pichia pastoris hosts. Loss of the C-teminal domain decreased the ability of the chitinase to bind and degrade insoluble substrates. Differences in optimal pH and temperature, thermostability, and ability to degrade certain oligosaccharide substrates were also observed between Bmchi and Bmchia dagger c. To determine whether these proteins could be used to increase virulence of entomopathogenic fungi, Bmchi and Bmchia dagger c were introduced into Beauveira bassiana under control of the A. nidulans gpdA constitutive promoter. Insect bioassays using WT and transformed B. bassiana strains revealed expression of Bmchi significantly improved fungal virulence compared to the WT parental strain, whereas expression of Bmchia dagger c in B. bassiana had only a nominal effect. These results indicate that the B. mori chitinase can be used to improved fungal efficiency but that the C-terminal domain is essential for this function.