粘质沙雷氏菌马来酸顺反异构酶表达纯化及酶学性质

Purification and Characterization of Maleate Cis-Trans Isomerase from Serratia marcescens

DOI：10.3969/j.issn.1673-1689.2014.11.013

中文关键词: 马来酸顺反异构酶 基因重组与表达 酶学性质 酶热稳定性

英文关键词: maleate cis-trans isomerase, gene recombinant and expression, enzymatic properties, thermostability

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中文摘要:

从粘质沙雷氏菌(Serratia marcescens)中扩增得到马来酸顺反异构酶基因。将该基因连接到pET24a载体上, 并在Escherichia coli BL21(DE3)中实现了成功表达。酶学性质研究表明, 重组酶的比酶活力为48.01 U/mg。其最适温度为37 ℃, 最适pH为8.4。在最适反应条件下, Km值为4.20 mmol/L, Vmax为1.27 mmol/(L·min), kcat为4.38 s-1, 催化效率kcat/Km为1.04 L/(mmol·s)。研究结果进一步显示, 马来酸顺反异构酶具备良好的热稳定性(55 ℃半衰期为1.5 h)及高达99%以上的转化率, 为马来酸顺反异构酶的进一步研究和工业应用制备高纯度的富马酸提供了理论基础和支持。

英文摘要:

The gene of maleate cis-trans isomerase(MaiA) from Serratia marcescens was cloned and successfully expressed in Escherichia coli BL21(DE3) and was purified. The specific activity of the purified enzyme was 48.01 U/mg. The optimum temperature was 37 ℃ and the optimum pH was 8.4. Under the optimum condition, the Km value was 4.2 mmol/L, the Vmax value was 1.27 mmol/(L·min), the kcat value was 4.38 s-1 and the catalytic efficiency(kcat/Km) was 1.04 L/(mmol·s). Furthermore, the research showed that the MaiA had high thermostability(t1/2=1.5 h). Meanwhile, maleate could almost be converted to fumarate completely by the recombinant MaiA with high efficiency up to 99%. The results in this study will be useful for further research and industrial applications of maleate cis-trans isomerase.

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