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当前位置: 首页 > 文章 > 谷氨酸棒杆菌乳酰谷胱甘肽裂合酶基因克隆、表达及活性分析 河北农业大学学报 2019 (2) 54-59
Position: Home > Articles > Gene cloning, expression and activity analysis of the lactoylglutathione lyase in Corynebacterium glutamicum Journal of Agricultural University of Hebei 2019 (2) 54-59

谷氨酸棒杆菌乳酰谷胱甘肽裂合酶基因克隆、表达及活性分析

作  者：
郝成伟;李贺丹;张献;徐大庆
单  位：
河北农业大学生命科学学院
关键词：
谷氨酸棒状杆菌;乳酰谷胱甘肽裂合酶;基因表达;酶活分析
摘  要：
乳酰谷胱甘肽裂合酶是生物体内降解丙酮醛的重要酶之一。食品级微生物谷氨酸棒杆菌ATCC13032基因组上的NCgl0106预测为乳酰谷胱甘肽裂合酶基因,但尚缺乏实验验证。本试验首先通过pCR技术扩增出预测的谷氨酸棒状杆菌ATCC13032乳酰谷胱甘肽裂合酶基因lac,并将之与表达载体pET-28a连接,转化BL21(DE3)感受态细胞,成功获得工程菌E.coli BL21(DE3)/pET-28a-lac。然后,使用IpTG诱导E.coli BL21(DE3)/pET-28a-lac表达重组蛋白Lac。SDS-PAGE试验结果表明:重组Lac蛋白在大肠杆菌进行了可溶性表达,其分子量约为14 kDa。最后对重组Lac蛋白进行了分离纯化、活性检测及酶学特性分析。应用His-tag纯化获得的重组蛋白溶液浓度为604.9μg/mL;活性检测显示重组Lac蛋白能催化丙酮醛和谷胱甘肽生成S-D-乳酰谷胱甘肽,表明谷氨酸棒杆菌lac基因是乳酰谷胱甘肽裂合酶基因;酶学特性分析表明重组乳酰谷胱甘肽裂合酶的最适反应温度为60℃,最适反应pH为7,金属离子K~+和Ca~(2+)对其活性略有增强作用,Ba~(2+)对酶活性几乎没有影响,Zn~(2+)、Mg~(2+)和Fe~(2+)对酶活性有一定的抑制作用,Co~(2+)和Mn~(2+)几乎完全抑制其活性,酶反应米氏常数K_m值为0.223 2 mmol/L,最大反应速率为0.025 mmol/(L·min)。
译  名：
Gene cloning, expression and activity analysis of the lactoylglutathione lyase in Corynebacterium glutamicum
作  者：
HAO Chengwei;LI Hedan;ZHANG Xian;XU Daqing;College of Life Sciences, Hebei Agricultural University;
关键词：
Corynebacterium glutamicum;;lactoylglutathione lyase;;gene expression;;enzymatic activity assay
摘  要：
Lactoylglutathione lyase is one of the enzymes degrading pyruvaldehyde. The NCgl0106 gene in the chromosome of C. glutamicum ATCC13032 was predicted as lactoylglutathione lyase gene, which is still not experimentally verified. In this study, firstly, the predicted lactoylglutathione lyase gene lac of C. glutamicum was amplified by PCR and inserted into the plasmid pET-28a. The recombinant plasmid pET-28a-lac was transformed into Escherichia coli BL21(DE3), resulting in the engineered strain E. coli BL21(DE3)/pET-28a-lac. Secondly, the recombinant target protein Lac was expressed in E. coli BL21(DE3)/pET-28a-lac by IPTG induction. The SDSPAGE results showed that the Lac protein was successfully expressed in the form of dissoluble protein in E.coli and its molecular weight is approximately14 kDa. Finally, the purification, and activity analysis and enzymatic characterizations test of the recombinant Lac protein were carried out. The Lac protein was successfully purified using His-tag and its solution concentration was 135.69 μg/mL. The activity analysis result showed the ability of the Lac protein to convert the substrates pyruvaldehyde and glutathione into the product lactoylglutathione,demonstrating that the NCgl0106 expressed protein is lactoylglutathione lyase. The enzymatic characterizations test showed that the optimum temperature of Lac was 60 ℃; the optimum pH was 7; the metal ions K~+ and Ca~(2+) had a slightly enhanced effect on Lac activity; the metal ion Ba~(2+) had little effect on enzyme activity; Zn~(2+), Mg~(2+) and Fe~(2+)had a certain inhibitory effect on enzyme activity and Mn~(2+) and Co~(2+) completely inhibited enzyme activity. The K_m value of the enzymatic reaction was 0.2232 mmol/L and its maximum reaction rate V_(max) was 0.025 mmol/(L·min).

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