费氏弧菌β-内切葡聚糖酶基因在大肠杆菌中的表达及酶学分析

以费氏弧菌（Vibrio fischeri）EM17基因组DNA为模板，通过PCR方法克隆了该菌的β-内切葡聚糖酶基因（GenBank Accession Number : EF533943）。将其插入到表达载体pGEX-6p-1中，转化大肠杆菌BL21（DE3），诱导表达。测序结果经DNAMAN软件分析，该基因与GeneBank中费氏弧菌 ES114基因组预测的β-内切葡聚糖酶基因同源性达到97.1％，但与GeneBank中其他β-内切葡聚糖酶基因同源性较低。酶学性质研究表明，该酶的最适反应温度为60℃，最适反应pH值为9.0。同时研究了6种金属离子对该酶活性的影响，结果显示该酶活性依赖金属离子的存在 Mg2+，Ca2+对酶有激活作用，Mn2+, Pb2+则严重抑制该酶的活性。

Expression of β-endoglucanase Gene from Vibrio fischeri in Escherichia coli and Its Enzymological Characterization

A gene encoding β-endoglucanase (EG) from Vibrio fischeri EM17 was amplified by PCR and inserted into E.coli expression plasmid pGEX-6p-1.The recombinant plasmid was transformed into E.coli BL21 and induced to express. Sequecing analysis showed that the sequence of β-endoglucanase gene fragment exhibited 97.1％ homology with Vibrio fischeri ES114’s ,while it was very low compared with other organisms’ .The optimal enzyme reaction temperature is 60℃, and the optimal pH is 9.0. The result of the effect of ion on enzyme activity showed that the enzyme activity relied on metal ion, Mg2+and Ca2+ showed a stimulating effect while Mn2+ and Pb2+ showed an strong inhibitory effect.