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p ET-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(Microbulbiferarenaceous)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。
[Background]β-glucan is a non-starch polysaccharide,widely existed in nature,especially in cell wall of cereal plants.β-glucanase can catalyzeβ-glucan intoβ-glucooligosaccharides,it plays an important role in food,feed and papermaking industry.[Objective]Gene ofβ-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]Theβ-1,3(4)-glucanase gene(MaGlu16 A)was cloned from the genomic DNA of Microbulbifer arenaceous and the recombinant plasmid(pET-28 a-MaGlu16 A)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 MaGlu16 A 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,MaGlu16 A showed broad substrate specificity.It could hydrolyze not only curdlan and laminarin but also barleyβ-glucan,lichenan,oatβ-glucan,and yeastβ-glucan,whose hydrolytic products were mainly glucose,disaccharide,trisaccharide and tetragasaccharide.[Conclusion]Cloning,expression and characterization ofβ-1,3(4)-glucanase from marine bacteria Microbulbifer arenaceous could provide a basis for the exploration ofβ-glucanase and preparation ofβ-glucooligosaccharides.
作者
马俊文
江正强
李晨霞
闫巧娟
MA Jun-Wen;JIANG Zheng-Qiang;LI Chen-Xia;YAN Qiao-Juan(Key Laboratory of Food Bioengineering(China National Light Industry),College of Engineering,China Agricultural University,Beijing 100083,China;College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2020年第7期2028-2039,共12页
Microbiology China
基金
国家自然科学基金(31630096)。
