D-阿洛酮糖3-差向异构酶的异源表达和酶学性质

Heterologous Expression and Enzymatic Characterization of D-Psicose 3-Epimerase

克隆了来源于Clostridium bolteae ATCC BAA-613的D-阿洛酮糖3-差向异构酶基因,利用重叠延伸PCR技术在Cb-dpe基因的上游加入了P43启动子,形成P43-Cb-dpe,再将P43-Cb-dpe连接到p MA5载体上构建出双启动子表达载体,并导入到Bacillus subtilis WB800中进行表达;与单启动子表达系统相比,双启动子表达载体能够显著提高Cb-dpe的表达量。对重组Cb-DPE酶进行了分离纯化和酶学性质的研究,结果表明:重组Cb-DPE的最适温度为55℃,最适pH为7.0,在温度30~40℃范围内和pH 6.5~7.5之间有良好的稳定性;Co^(2+)、Mn^(2+)可显著增强酶活;D-阿洛酮糖为底物时,K_m为26.68 mmol/L,小于果糖的61.80 mmol/L,说明该酶对D-阿洛酮糖的亲和性比对D-果糖的高。而在动力学参数方面,以D-阿洛酮糖为底物对应的催化效率K_(cat)/K_m为95.8 L/(mmol·min),大于以果糖作为底物时的54.1 L/(mmol·min)。

The DPE gene from Clostridium bolteae ATCC BAA-613 was cloned and expressed in Bacillus subtilis WB800. We inserted a P43 promoter directly upstream the DPE geneby virtue of overlap extension PCR, which forming P43-Cb-dpe, and then ligated it into pMA5 shuttle vector thus constructing the pMA5-P43-Cb-dpe expression vector. The recombinant plasmid was imported into Bacillus subtilis WB800 for expression. Compared with single promoter expression system, pMA5-P43-Cb-dpe has a palpable promotion of expression level. The expressed recombinant enzyme was purified andits enzymatic properties were studied. The optimum temperature and pH for recombinant enzyme were 55 ℃ and pH 7.0,respectively. It was highly stable at 30-40 ℃ and pH 6.5 -7.5. Co^2＋ and Mn^2＋ can significantly enhance enzyme activity. The recombinant enzyme＇s affinity for D-psicose was high than D-frutcose. Meanwhile,in terms of the kinetic parameters, the catalytic efficiency Kcat/Km is greater when use D-psicose as substrate.