Yan R T, Chen J S
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Appl Environ Microbiol. 1990 Sep;56(9):2591-9. doi: 10.1128/aem.56.9.2591-2599.1990.
Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.
乙醛和丁醛是产溶剂梭菌生产乙醇和1-丁醇过程中醇脱氢酶的底物。一种辅酶A（CoA）酰化醛脱氢酶（ALDH），它也能将酰基辅酶A转化为醛和辅酶A，已在厌氧条件下从拜氏梭菌NRRL B592中纯化出来。该ALDH的天然分子量（Mr）为100,000，亚基分子量为55,000，表明ALDH是二聚体。纯化的ALDH不具有醇脱氢酶活性。以乙醛和丁醛作为替代底物测得的活性被共纯化，这表明相同的ALDH可以催化生成用于乙醇和丁醇生产的两种醛。基于乙酰辅酶A和丁酰辅酶A的Km和Vmax值，ALDH对丁醛生产的效果比对乙醛生产的效果更好。ALDH可以使用NAD(H)或NADP(H)作为辅酶，但NAD(H)的Km远低于NADP(H)。动力学数据表明该反应存在乒乓机制。ALDH在Tris缓冲液中比在磷酸盐缓冲液中更稳定。正向反应（生理方向；醛形成）的表观最适pH在6.5至7之间，反向反应（酰基辅酶A形成）的表观最适pH为9.5或更高。NAD(H)/NADP(H)连接活性的比例随pH降低而增加。ALDH对O2敏感，但二硫苏糖醇可以保护其免受O2失活。在测定前，通过在有或没有二硫苏糖醇的情况下将酶与辅酶A一起孵育，可以使O2失活的酶重新激活。
