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

DOI：10.3969/j.issn.1673-1689.2018.03.010

中文关键词: D-阿洛酮糖3-差向异构酶 D-阿洛酮糖 双启动子 酶学性质

英文关键词: D-psicose 3-epimerase,D-psicose,double promoters,enzymatic properties

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中文摘要:

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pMA5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；Co2+、Mn2+可显著增强酶活；D-阿洛酮糖为底物时，Km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Kcat/Km为95.8 L/（mmol·min），大于以果糖作为底物时的54.1 L/（mmol·min）。

英文摘要:

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. Co2+ and Mn2+ 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.

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