一种耐热偏酸性β-甘露聚糖酶基因克隆与高效表达

Cloning and efficient expression of a thermostable and weak acidic β-mannanase gene

【目的】克隆半纤维素降解高效菌株Bacillus subtilis BE-91的甘露聚糖酶基因并进行原核表达,对表达产物进行酶学性质研究。【方法】采用PCR扩增法从B.subtilis BE-91菌株中克隆β-甘露聚糖酶基因,分别连接到p EASY-E1和p ET28a载体,导入Escherichia coli BL21(DE3)进行诱导表达。用DNS法对工程菌株的胞内和胞外β-甘露聚糖酶进行定量分析,选取胞外甘露聚糖酶活力高的组分进行酶学性质研究。【结果】从B.subtilis BE-91菌株中克隆的β-甘露聚糖酶基因(Gen Bank登录号:KP277209)在E.coli中获得高效表达,工程菌株p EASY-man/BL产胞外β-甘露聚糖酶的活性可达229.1 IU/m L;该基因序列全长960 bp,包含319个氨基酸的编码序列和一个终止密码子;表达产物的最适反应温度为65°C,最适反应p H为6.0,属于耐热偏酸性β-甘露聚糖酶;该酶稳定温度≤65°C,稳定p H为4.5-7.0;1 mmol/L的Cu2+、Mn2+、Zn2+、Ca2+对该酶有激活作用,而Ba2+和Pb2+有强烈抑制作用。【结论】B.subtilis BE-91拥有珍贵的β-甘露聚糖酶基因资源,其胞外表达产物的耐热偏酸性酶学性质在开发饲料添加剂方面具有潜在的应用前景。

[Objective] The β-mannanase gene（man） from Bacillus subtilis BE-91 which is an efficient strain for hemi-cellulose degradation was cloned and expressed in Escherichia coli BL21（DE3）, then the enzymatic properties of the β-mannanase（Man） were studied. [Methods] The man was amplified from B. subtilis BE-91 by PCR, respectively linked to p EASY-E1 and p ET28 a, and finally expressed in E. coli BL21（DE3）. After comparing the extracellular and intracellular β-mannanase activities from the gene engineering strains, the β-mannanase with highest activity was selected to study the enzymatic properties fully. [Results] The man A（Gen Bank： KP277209） was 960 bp in length, including a termination codon and encoded 319 amino acids. The highest activity of the extracellular β-mannanase from p EASY-man/BL strain was 229.1 IU/m L. The optimal temperature and p H of the β-mannanase were 65 °C and 6.0. The catalytic activity of the enzyme was stable at no more than 65 °C and p H 4.5-7.0 after being incubated for 30 min. It was promoted by Cu2＋, Mn2＋, Zn2＋and Ca2＋, while inhibited seriously by Ba2＋ and Pb2＋ at concentration of 1 mmol/L. [Conclusion] A precious β-mannanase gene has been excavated from B. subtilis BE-91, and its expression product with properties of thermostability and weak acidity may be available for feed additive.