解淀粉芽孢杆菌HZ-1510壳聚糖酶基因的克隆、重组表达及其活性

摘要

To study the hydrolysis activity of chitosanase, in this report, the coding sequence of chitosanse from Bacillus amyloliquefaciens HZ-1510 was cloned, and glutathione S-transferase (GST) fused-chitosanase was expressed and purified fromEscherichia coli. Furthermore, we analysed the signal peptide, amino acid sequence and its three-dimensional structure. In the end, its chitosan hydrolysis activity was measured. The chitosanase gene consisted of an open reading frame of 837 bp which encoded a protein of 279 amino acids with predicted molecular weight of 31.45 ku. The protein contained a signal peptide with a cleavage site located between the 36th and 37th amino acids. The deduced amino acid sequence homology analysis of the chitosanase revealed that it belonged to GH46 family. The recombinant chitosanase has been purified to homogeneity by using only GST column chromatography. The optimal pH and temperature for the chitosan hydrolysis activity was 5.5 and 55 °C, respectively. The enzyme activity was increased in the presence of Mn2+, Ca2+ and Mg2+. However, Fe3+, Ag+, Cu2+, Ba2+ and K+ could inhibit its activity. Furthermore, its activity could be increased in the presence of 0.1 mmol/L Zn2+ while it was inhibited by 2.0 mmol/L Zn2+. This study explored the optimal condition for hydrolytic activity of chitosanase and established a theoretical basis for its industrial application.%为了研究壳聚糖酶的水解活性,实验进行了解淀粉芽孢杆菌HZ-1510壳聚糖酶基因编码序列的克隆,并构建了谷胱甘肽转移酶(GST)-壳聚糖酶融合蛋白表达质粒,在大肠杆菌中表达后提取、纯化得到重组蛋白,并通过生物信息学对该蛋白的信号肽、三维结构等进行了分析,最后以胶态壳聚糖溶液为底物研究了该重组壳聚糖酶的活性.结果显示,该壳聚糖酶基因的ORF长为837 bp,编码279个氨基酸,分子量为31.45 ku.在其氨基末端具有信号肽,切割点位于36和37位氨基酸之间.氨基酸序列同源性分析表明该壳聚糖酶属于GH46家族糖苷水解酶.酶的水解活性最适温度约为55°C,最适pH为5.5.而金属离子Fe3+、Ag+、Cu2+、Ba2+和K+对其水解活性都起抑制作用,但是Mn2+、Ca2+和Mg2+对其活性起增强作用.0.1 mmol/L的Zn2+对壳聚糖酶的活性起增强作用,2.0 mmol/L的Zn2+对壳聚糖酶的活性起抑制作用.本实验研究了解淀粉芽孢杆菌HZ-1510壳聚糖酶在不同条件下的水解活性,为壳聚糖酶的工业应用奠定了理论基础.