화학공학소재연구정보센터
Journal of Fermentation and Bioengineering, Vol.86, No.3, 290-295, 1998
Cloning, expression and nucleotide sequence of the L-2,3-butanediol dehydrogenase gene from Brevibacterium saccharolyticum C-1012
A 3-kbp DNA fragment including the L-2,3-butanediol dehydrogenase (L-BDH) gene (budC) from the chromosomal DNA of Brevibacterium saccharolyticum C-1012 was cloned in Escherichia coli JM109 after its insertion into pBluescript II SK+, and the resulting plasmid was named pLBD-SK. The budC had an open reading frame consisting of 774 bp and encoded 258 amino acids. It was not included in a 2,3-butanediol operon such as is seen in the case of the meso-BDH gene (budC) of Klebsiella pneumoniae. For the expression of the budC, the deletion plasmid pLBD2-119 was prepared from pLBD-SK. E. coli JM109/pLBD2-119 had higher L-BDH activity than that of Br, saccharolyticum C-1012. The L-BDH appeared as two bands on disc-PAGE, Isopropyl-beta-D-thiogalacto-pyranoside (IPTG) influenced the quantity ratio of the electrophoretic isoenzymes of L-BDH from E. coli JM109/pLBD2-119; that is, a higher relative mobility band with weak substrate specificity was abundantly produced by IPTG. The BDH was considered to belong to the short-chain dehydrogenase/reductase (SDR) family on the basis of the following distinctive features : it possessed two conservative sequences GXXXGXG and YXXXK, and it consisted of about 250 amino acids. As a result of a phylogenetic analysis of SDR family enzymes, the BDHs were considered to comprise a cluster independent from the other SDR enzymes.