纤维微菌XM-8木聚糖酶基因克隆及酶学性质

Gene Cloning and Enzymology Characteristics Analysis of a Xylanase Gene from Cellulosimicrobium XM-8

关键词:  纤维微菌  木聚糖酶  共表达  水解

Key words:  Cellulosimicrobium  xylanase  coexpression  hydrolase

摘要:

[目的]为获得可应用于木聚糖水解的酶资源,希望通过筛选分离得到能够水解木聚糖的木聚糖酶产生菌,克隆表达木聚糖酶基因并研究其酶学性质。[方法]从环境中筛选分离出可水解木聚糖的菌株,利用16S rDNA对其进行分子鉴定。扩增其木聚糖酶基因,以pET22b (+)为表达载体,构建共表达重组质粒,转化Escherichia coli BL21(DE3)进行异源表达,并对重组酶进行酶学性质研究。[结果]经16S rDNA鉴定该菌株为纤维微菌。通过PCR成功克隆到该菌的木聚糖酶基因(xyn-8a),并构建共表达质粒pET22b-xyn-8a,实现木聚糖酶Xyn-8a的活性表达。酶学性质研究表明Xyn-8a最适反应温度为60℃,最适反应pH值为6.0,只对木聚糖底物有活性;HPLC分析其水解产物以木二糖为主,还有少量的木糖和木三糖。[结论]XYN-8A在pH值为6的条件下具有较高活力,且可以催化水解反应,在生产低聚木糖方面具有一定的应用价值。

Abstract:

[Objective] In order to obtain the enzyme resources which can be applied to the hydrolysis of xylan, xylanase producing strains are isolated and purified by screening, and the xylanase gene is cloned and characterized in this study.[Methods] The hydrolyzable xylan are isolated from the environment and molecular identified by 16S rDNA.The xylanase gene is amplified and co-expressed with pET22b (+) vector.The recombinant plasmid is transformed into Escherichia coli BL21 (DE3) for heterologous expression and the enzymatic properties of the recombinant enzyme are studied.[Results] The strain is identified as the genus of Cellulosimicrobium by 16S rDNA sequence.And the xylanase gene (xyn-8a) is cloned. The coexpression recombinant plasmid of pET22b-xyn-8a is successfully constructed and expressed in Escherichia coli BL21 (DE3).The maximum activity of Xyn-8a is obtained at 60℃, pH 6.0.It is only active on xylan substrates.Through HPLC analyzing its hydrolyzate is dominated by xylobiose, it also contains a small amount of xylose and xylotriose.[Conclusion] Xyn-8a has higher activity in pH 6, and can catalyze hydrolysis reaction.It has a certain application value in the production of xylo-oligosaccharides.