知识与财富的链接
【目的】通过两端融合表达几丁质结合结构域来提高几丁质酶的活性和生物防治植物病原真菌能力。【方法】以苜蓿链霉菌（Streptomyces alfalae）ACCC40021中唯一的GH19家族几丁质酶为模板,构建两端融合表达几丁质结合结构域的几丁质酶,并进行原核表达;利用3,5-二硝基水杨酸法（DNS）测定几丁质酶活。【结果】成功构建了CatDChiB （催化结构域）、rChiB （含N-端几丁质结合结构域）、DChBDChiB（含两端几丁质结合结构域）三种形式的酶,并在大肠杆菌中得到了高效表达;与CatDChiB和rChiB相比,DChBDChiB显著地提高了对α-几丁质、胶体几丁质和黑曲霉几丁质的结合能力和活性;同时增强了其对病原真菌长枝木霉的抑制作用。【结论】两端融合表达几丁质结合结构域是简单有效的提高几丁质酶活性及抗真菌活性的策略。
[Objective] To develop a simple method to improve the enzymatic and antifungal activity of chitinase by fusion of chitin binding domain at both termini. [Methods] The chimeric and truncated chitinases were constructed using the unique glycoside hydrolase family 19 chitinase in Streptomyces alfalae ACCC40021 as template, and expressed in Escherichia coli. The 3,5-dinitrosalicylic acid (DNS) method was used to determine the enzyme activity with colloid chitin as the substrate. [Results] CatDChiB (catalytic domain), rChiB (N-terminal chitin-binding domain) and DChBDChiB (double chitin-binding domain) were successfully constructed and expressed in E. coli BL21(DE3). Compared to CatDChiB and rChiB, DChBDChiB improved the binding ability and activity towards colloidal chitin, α-chitin and chitin from Aspergillus niger. Furthermore, antifungal activity was enhanced against plant pathogenic fungus Trichoderma longibranchiatum. [Conclusion] A simple, feasible and efficient method was developed to improve the enzymatic and antifungal activity of chitinase by fusion of chitin binding domain on both C-and N-terminus of protein.
