拜氏梭菌(Clostridium beijerinckii)NRRL-B593在三种不同培养基中乙醇脱氢酶活力的测定

Determination of Alcohol Dehydrogenase Activities in Clostridium beijerinckii grown on various substrates

拜氏梭菌(Clostridium beijerinckii〗)NRRL-B593在葡萄糖基质中可以代谢产生丁醇、乙醇、异丙醇。乙醇脱氢酶(ADH)是产生醇类的关键酶,比较了拜氏梭菌(C.beijerinckii)NRRL-B593在葡萄糖基质与氨基酸基质中ADH活性,为进一步研究该菌株的代谢及表达调控提供理论基础。首先通过监测C.beijerinckii NRRL-B593在不同培养基中D_(600)值,绘制生长曲线,后在8000 r/min,10min下收集对数期与稳定期的细胞,利用厌氧型细胞破碎仪French Press获得无细胞抽提物(CFE),最后通过Bradford法测量蛋白质含量,在厌氧条件下利用丙酮为底物,NADPH依赖型的氧化反应测其酶活力。结果显示,稳定期的ADH酶活力远远高于对数期酶活力高,氨基酸培养基中的ADH酶活力高于葡萄糖培养基。

Clostridium beijerinckii NRRL-B593 grows on glucose and is capable of producing butanol,ethanol and isopropanol. Alcohol dehydrogenase（ ADH） is a key enzyme catalyzing the production of alcohols. This article compared its ADH activities when it would grow on carbohydrates and peptides,and did the foundation for futher studying AHD. Clostridium beijerinckii NRRL-B593 growth was monitored using optical density at 600 nm. The growth curves were obtained using different substrates. Cells grown at log phase and stationary phase were harvested using centrifugation（ 8000 × g,10 mins）,and cell free extract（ CFE） was prepared using a French Press. Protein concentration was determined using the Bio-Rad assay. The ADH activity was measured under anaerobic conditions following the acetone-dependent oxidation of NADPH. ADH activity in the CFE from C. beijerinckii NRRL-B593 cells grown on tryptone was higher than that from the cells grown on glucose.