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首页> 外文期刊>African Journal of Biotechnology >Expression, purification and characterization of Oryza sativa L. NAD-malic enzyme in Escherichia coli
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Expression, purification and characterization of Oryza sativa L. NAD-malic enzyme in Escherichia coli

机译：水稻稻NAD-苹果酸酶在大肠杆菌中的表达，纯化和鉴定

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摘要

The cDNA fragment of?a rice?NAD-malic enzyme?(OsNAD-ME1)?was cloned and constructed into expression vector (pGEX-6p-3).?OsNAD-ME1?was successfully expressed as a GST fusion protein in?Escherichia coli?BL21.?The optimal concentration of IPTG for inducement was 1?mmol/L?and the optimal?culturetemperature was 30°C. The fusion protein was purified by using affinity chromatography with a glutathione sepharose 4B column. After enzymatic cleavage of GST tag, the?OsNAD-ME1?recombinant protein was collected for studying its kinetic properties. The optimum pH and temperature for catalytic reaction?ofOsNAD-ME1?were pH 6.4 and 35°C, respectively. The?kcat?value determined at pH 6.4 was?36.38?s-1?and the?Km?values?for NAD+?and malate were 0.10 and 15.98mmol/L, respectively. The maximum activity of?OsNAD-ME1?using NADP+?as coenzyme?was 64.47% of that using NAD+?as coenzyme.
机译：克隆了水稻“ NAD-苹果酸酶”（OsNAD-ME1）的cDNA片段，并将其构建到表达载体（pGEX-6p-3）中。“ OsNAD-ME1”在大肠杆菌中成功表达为GST融合蛋白。大肠埃希菌BL21诱导的最佳IPTG浓度为1μmmol/ L，最佳培养温度为30°C。通过使用具有谷胱甘肽琼脂糖4B柱的亲和色谱法纯化融合蛋白。酶切GST标签后，收集ΔOsNAD-ME1Δ重组蛋白以研究其动力学性质。 OsNAD-ME1催化反应的最佳pH和温度分别为6.4和35°C。在pH 6.4下测得的“ kcat”值是“ 36.38” s-1”，而NAD +”和苹果酸的“ Km”值分别是0.10和15.98mmol / L。以NADP +为辅酶的“ OsNAD-ME1”的最大活性为以NAD +为辅酶的最大活性的64.47％。

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