沙质微泡菌β-1,3(4)-葡聚糖酶的克隆表达及酶学性质

Cloning, expression and characterization of an β-1,3(4)-glucanase from Microbulbifer arenaceous

摘要:

【背景】β-葡聚糖是自然界中广泛存在的非淀粉多糖，是谷类植物细胞壁的主要成分。β-葡聚糖酶能够水解β-葡聚糖生成低聚合度的寡糖，在食品、饲料、造纸等领域发挥着重要的作用。【目的】从海洋细菌沙质微泡菌(Microbulbifer arenaceous)中克隆到一个β-1,3(4)-葡聚糖酶基因，在大肠杆菌中可溶表达，研究其相关酶学性质。【方法】以沙质微泡菌(Microbulbifer arenaceous)基因组DNA为模板，克隆一个β-1,3(4)-葡聚糖酶基因(MaGlu16A)，构建重组表达载体pET-28a-MaGlu16A并在大肠杆菌BL21(DE3)中诱导表达，通过Ni-NTA亲和层析纯化后进行酶学性质研究。【结果】MaGlu16A的最适pH和最适温度分别为pH 6.0和40 °C，在pH 5.0?10.5和35 °C以下稳定。对EDTA具有较高的抵抗性，在1 mmol/L和10 mmol/L EDTA浓度下仍保持99.3%和82.5%的酶活力。该酶能够有效水解可得然多糖、昆布多糖、大麦葡聚糖、地衣多糖、燕麦葡聚糖和酵母葡聚糖，水解产物主要为葡萄糖、二糖、三糖和四糖。【结论】海洋细菌沙质微泡菌(Microbulbifer arenaceous)来源β-1,3(4)-葡聚糖酶的克隆表达及酶学性质的测定为β-葡聚糖酶的挖掘及β-葡寡糖的制备奠定了基础。

Abstract:

[Background] β-glucan is a non-starch polysaccharide, widely existed in nature, especially in cell wall of cereal plants. β-glucanase can catalyze β-glucan into β-glucooligosaccharides, it plays an important role in food, feed and papermaking industry. [Objective] gene of β-1,3(4)-glucanase from marine bacteria Microbulbifer arenaceous was cloned and expressed in Escherichia coli, and its enzymatic characteristics and hydrolysis properties were studied. [Methods] The β-1,3(4)-glucanase gene (MaGlu16A) was cloned from the genomic DNA of Microbulbifer arenaceous and the recombinant plasmid (pET-28a-MaGlu16A) was constructed. The recombination strain was successfully expressed in E. coli BL21(DE3), and purified by Ni-NTA affinity chromatography, then enzymatic characteristics was studied. [Results] The optimal pH of MaGlu16A was 6.0 and optimal temperature was 40 °C. It was stable between pH 5.0 and 10.5, and below 35 °C. The enzyme showed obvious resistance to EDTA, and it could maintain 99.3% and 82.5% activity at the concentration of 1 mmol/L and 10 mmol/L. In addition, MaGlu16A showed broad substrate specificity. It could hydrolyze not only curdlan and laminarin but also barley β-glucan, lichenan, oat β-glucan, and yeast β-glucan, whose hydrolytic products were mainly glucose, disaccharide, trisaccharide and tetragasaccharide. [Conclusion] Cloning, expression and characterization of β-1,3(4)-glucanase from marine bacteria Microbulbifer arenaceous could provide a basis for the exploration of β-glucanase and preparation of β-glucooligosaccharides.