Abstract

A novel chi19 gene encoding the chitinase, belonging to the glycosyl hydrolase family 19 from the predatory plant Drosera capensis is cloned and expressed in the E. coli bacterial system. The amino acid sequence of the translated enzyme consists of 325 a.a. divided into four functional parts: the N-terminal signal sequence and of the catalytic and chitin-binding domains that were linked by the –S–P- linker. The chi19 gene is expressed in two forms: with and without a chitin-binding domain. Refolding is carried out and homogeneous soluble forms are obtained for both variants of the enzyme. The chitinases obtained exhibit predominantly specific activity towards crystalline chitin, with the optimal pH level ranging from 5.0 to 5.5 and the optimum temperature in the range of 52 to 55°C for both forms of the enzyme. The catalytic activity of the full-sized form of chitinase was 360 U/g with a 64% decrease of the catalytic activity for the form with the chitin-binding domain removed.

中文翻译：

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食肉植物鸭茅的新型几丁质酶的克隆与表达

摘要

克隆一种新的编码几丁质酶的chi19基因，该酶属于捕食性植物Drosera capensis的糖基水解酶家族19，并在大肠杆菌中表达。翻译后的酶的氨基酸序列由325个氨基酸组成，分为四个功能部分：N末端信号序列以及由–S–P-接头连接的催化和几丁质结合结构域。该chi19基因以两种形式表达：有和没有甲壳质结合结构域。对酶的两个变体进行重折叠并获得均质的可溶形式。对于两种形式的酶，所获得的几丁质酶主要表现出对结晶甲壳质的比活性，最佳pH水平为5.0至5.5，最佳温度为52至55℃。完整尺寸的几丁质酶的催化活性为360 U / g，对于去除了几丁质结合域的形式，其催化活性降低了64％。