Purification and studies of two butanol(ethanol) dehydrogenases and the effects of rifampicin and chloramphenicol on other enzymes important in the production of butyrate and butanol in Clostridium acetobutylicum ATCC 824.

摘要

Two NADH dependent butanol(ethanol) dehydrogenases from Clostridium acetobutylicum have been purified to near homogeneity. Either of these two dehydrogenases could be part of the pathway for the production of butanola and are thus important for an understanding of the metabolism involved in butanol production. Another butanol dehydrogenase was detected that was largely dependent on NADPH. Studies were also carried out on the effects of rifampicin and chloramphenicol on the metabolism of C. acetobutylicum.; Butanol dehydrogenases I and II are composed of two subunits of about 42 kD in weight, with a native molecular weight of 80 kD. Both enzymes were purified with Zn{dollar}sp{lcub}2+{rcub}{dollar} in the buffer to improve the recovery of enzyme activity. The enzymes were more active at around pH 6.0, within the physiological pH range of 5.6-6.8 as reported for C. acetobutylicum. Both enzymes were substantially more active in the normal metabolic direction (butyraldehyde to butanol), and the kinetics of this reaction were studied in this direction. Both enzymes were determined to have ordered bi bi kinetic binding mechanisms. This mechanism was determined through substrate inhibition analysis with butyraldehyde for BDH I and through competitive and product inhibition studies for BDH II. The reactivity of the enzymes towards other substrates was also determined; the rate of the reduction of aldehydes appears to increase with increasing chain length for both butanol dehydrogenase I and II, none of the ketones studied acted as substrates. The most important difference is that BDH I was substantially more reactive towards acetaldehyde than BDH II. The effect of various metabolites, such as ATP and CoA, was also determined and the results were similar for each enzyme. The amino acid sequences and pI's were determined for both BDH I and II and slight differences were found.; The effect of the addition of either rifampicin or chloramphenicol to growing cultures was studied for the products: butyrate, acetate, butanol, and acetone, and for the enzymes: phosphotransbutyrylase, butyrate kinase, coenzyme A transferase, butyraldehyde dehydrogenase, and butanol dehydrogenase (both NADH and NADPH dependent). The results demonstrated that phosphotransbutyrylase and butyrate kinase are constitutive and stable in vivo. CoA transferase and the butanol dehydrogenases were all induced/derepressed and were also stable in vivo. Butyraldehyde dehydrogenase was induced/derepressed but was highly unstable in vivo. The instability of butyraldehyde dehydrogenase coupled with the location of butyraldehyde dehydrogenase as a branch point in the butanol pathway, suggests that butyraldehyde dehydrogenase is highly important in regulating the flow of butyryl-CoA and therefore in regulating the switch from butyrate to butanol production.