乙酰辅酶A合成酶的酶学性质及其在甲羟戊酸合成中的应用  

Enzymatic characterization of acetyl CoA synthetase and its application in biosynthesis of mevalonate

作　　者：胡红[1,2] 张汝兵[2] 秦杰明[1] 咸漠[2] 

机构地区：[1]长春理工大学材料科学与工程学院,长春130000 [2]中国科学院青岛生物能源与过程研究所,中国科学院生物基材料重点实验室,青岛266101

出　　处：《应用与环境生物学报》2016年第3期357-362,共6页Chinese Journal of Applied and Environmental Biology

基　　金：山东省自然科学基金项目(ZR 2015PB013);中国科学院重点部署项目(KGZD-EW-606-1-3)资助

摘　　要：乙酰辅酶A合成酶(ACS)在微生物体内可直接将乙酸转化为乙酰辅酶A,乙酰辅酶A再进入微生物的碳代谢循环过程.为提高乙酸的利用效率,筛选催化活性较高的乙酰辅酶A合成酶,从大肠杆菌(Escherichia coli K-12)和巴氏醋酸杆菌(Acetobacter pasteurianus ATCC 33445)中扩增得到3个不同的乙酰辅酶A合成酶基因,将这3个基因分别连接到p ET-28a(+)载体上,Western Blot分析表明,3个基因均在E.coli BL21(DE3)中成功表达.对3个不同来源的乙酰辅酶A合成酶的酶活性进行比较,发现来自于巴氏醋酸杆菌的乙酰辅酶A合成酶(ACS2)具有较高的催化活力;其最适温度37℃,最适pH在7-8之间;在最适反应条件下,K_m为1.16 mmol/L,最大反应速度(V_(max))为20.45 mmol L^(-1) min^(-1).将ACS2应用到以乙酸为底物生物合成甲羟戊酸过程中,当过表达ACS2时,单批补料发酵条件下,甲羟戊酸产量达到6.59g/L.本研究筛选到1种催化活性较高的乙酰辅酶A合成酶,显著提高了甲羟戊酸的生物合成,可为乙酰辅酶A合成酶的进一步研究和应用提供理论支持.(图8表1参18)Acetyl CoA synthetase （ACS） widely distributed in microorganisms can directly catalyze acetic acid to acetyl CoA, which is then metabolized in microbial carbon cycle. In this study, we cloned three different genes of acetyl CoA synthetases from Escherichia coli K-12 and Acetobacterpasteurianus ATCC 33445, and then inserted them into pET-28a（＋） vector. Western Blot analysis showed that the three ACS genes were successfully expressed in E. coli BL21（DE3）. Activity analysis showed that ACS2 from A. pasteurianus had the highest catalytic activity, so its enzymatic characteristics were further studied. The optimum temperature and pH of ACS2 were 37 ~C and 7-8 respectively. Under the optimum reaction conditions, the Km was 1.16 mmol/L and the maximum reaction rate was 20.45 mmol L＂~ rain-l. The results showed that application of overexpressed ACS2 to biosynthesis of mevalonate from acetic acid under fed-batch conditions could yield mevalonate at 6.59 g/L. This result provides theoretical support for further research and application of acetyl CoA synthetase.

关 键 词：乙酰辅酶合成酶 巴氏醋酸杆菌 酶学性质 乙酸 甲羟戊酸 

分 类 号：Q55[生物学—生物化学]