| 초록 |
Megasphaera nov. sp. BS-4 producing more 10 g/L of hexanoic acid was isolated from cow rumen. In order to establish the metabolic pathway for hexanoic acid production of Megasphaera nov. sp. BS-4, the full genome of Megasphaera nov. sp. BS-4 was analyzed and annotated through genome library. Five genes related to carbon chain elongation were predicted and selected. The predicted enzymes were acetyl-CoA acetyltransferase, 3-hydroxy-butyryl-CoA dehydrogenase, 3-hydroxy-butyryl CoA dehydratase, butyryl-CoA dehydrogenase and Acyl-CoA hydrolase. In order to confirm activities of the enzymes, enzymes were expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA column. As substrates, 0.5 mM acetyl CoA and 0.5 mM butyryl CoA were used and 0.2 mM NADH was added as a coenzyme. The enzymes were added into reactants step by step and 100 ul of reactant was sampled. The metabolites by sequential enzyme reaction were analyzed using HPLC-MS. |
