瑞氏木霉内切葡聚糖酶II在大肠杆菌中的重组表达及重组酶性质测定

Expression of Recombinant Trichoderma Reesei Endoglucanase II in Escherichia coli and Properties of the Recombinant Enzyme

将不含信号肽的瑞氏木霉内切葡聚糖酶II(Cel5A)的cDNA克隆到pET-28a(+)表达质粒上,与其N末端6个组氨酸标签序列融合,构建成pET-28a(+)-egl2表达质粒,在大肠杆菌BL21(DE3)中诱导表达.利用低温诱导策略,成功表达出活性重组蛋白.Western blot结果显示重组Cel5A相对分子分量大约为43000.进一步对重组Cel5A进行Ni-NTA亲和层析柱纯化并对其进行酶学性质测定,结果显示以羧甲基纤维素钠为作用底物时重组酶最适作用pH为4.5,最适作用温度为60℃;重组酶热稳定性较好,在65℃及以下保温1.5h,活性仍相当稳定.Mn2+对Cel5A的酶活有促进作用,Fe3+和Cu2+有抑制作用,其它金属离子和EDTA对酶活没有明显影响.底物特异性研究表明,重组Cel5A只能水解混合键葡聚糖和羧甲基纤维素钠,对混合键葡聚糖的水解能力是其对羧甲基纤维素钠水解能力的6.7倍,但对微晶纤维素、Glass microfiber filters、木聚糖、阿拉伯木聚糖和木葡聚糖没有水解作用.

Cel5A of Trichoderma reesei was fused with histidine（6） tag and cloned in pET-28a（＋） plasmid. With a low temperature induction strategy,the enzyme was successfully expressed in E. coli host. Western blot analysis showed that the recombinant Cel5A had a molecular weight of 43 000. The recombinant Cel5A had maximal activities at pH 4.5,60℃ and was stable when it was heated for 1.5 h at 65℃. The enzymatic activity was enhanced by 1 mmol/L Mn 2＋ and inhibited by Fe 3＋ and Cu 2＋ while other metal ions and EDTA had no effect. The recombinant Cel5A was able to hydrolyze β-glucan and CMC-Na,but unable to hydrolyze Avicel,Glass microfiber filters,Xylan,Arabiroxylan and Xyloglucan. The activity of Cel5A to digest β-glucan is 6.7-fold higher than to digest CMC-Na.