解淀粉芽孢杆菌HZ-1510壳聚糖酶基因的克

发布时间：2018-02-28 13:11

本文关键词： 解淀粉芽孢杆菌壳聚糖酶基因克隆蛋白表达酶活性　出处：《水产学报》2017年10期 　论文类型：期刊论文

【摘要】：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+、Cu~(2+)、Ba~(2+)和K+对其水解活性都起抑制作用,但是Mn~(2+)、Ca~(2+)和Mg~(2+)对其活性起增强作用。0.1 mmol/L的Zn~(2+)对壳聚糖酶的活性起增强作用,2.0 mmol/L的Zn~(2+)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。
[Abstract]:In order to study the hydrolysis activity of chitosanase, the HZ-1510 chitosanase gene encoding sequence of Bacillus amylum was cloned and the expression plasmid of glutathione transferase GST-chitosan fusion protein was constructed. The recombinant protein was extracted and purified from E. coli, and its signal peptide and three-dimensional structure were analyzed by bioinformatics. Finally, the activity of the recombinant chitosanase was studied using colloidal chitosan solution as the substrate. The results showed that the ORF length of the chitinase gene was 837bp, encoding 279 amino acids, and its molecular weight was 31.45 ku.There was a signal peptide at the amino end of the chitosanase gene. The amino acid sequence homology analysis showed that the chitosanase belongs to the GH46 family glycoside hydrolase. The optimum hydrolysis temperature of the enzyme is about 55 掳C and the optimum pH is 5.5. And K both inhibit its hydrolysis activity, However, Mn~(2 and Mg~(2) enhanced its activity. Zn~(2 (0.1 mmol/L) enhanced the activity of chitinase. Zn~(2 (2.0 mmol/L) inhibited the activity of chitinase. This study was conducted to investigate the effect of HZ-1510 chitosan on the activity of Bacillus amylococcus HZ-1510. The hydrolytic activity of glycolase under different conditions, It lays a theoretical foundation for the industrial application of chitosanase.
【作者单位】：华中农业大学水产学院;华中农业大学食品科技学院;仲恺农业工程学院动物科技学院;
【基金】：湖北省科技支撑计划(2015BBA228) 武汉市科技局基金(2016020101010089) 广东省海洋与渔业局基金(2015-033)~~
【分类号】：Q78;Q936

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