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-B DH) gene (budC) from the chromosomal DNA of Brevibacterium saccharolyt icum C-1012 was cloned in Escherichia coli JM109 after its insertion i nto pBluescript II SK+, and the resulting plasmid was named pLBD-SK. T he 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 i s seen in the case of the meso-BDH gene (budC) of Klebsiella pneumonia e. 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 activi ty than that of Br, saccharolyticum C-1012. The L-BDH appeared as two bands on disc-PAGE, Isopropyl-beta-D-thiogalacto-pyranoside (IPTG) inf luenced 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/reductas e (SDR) family on the basis of the following distinctive features: it possessed two conservative sequences GXXXGXG and YXXXK, and it consist ed of about 250 amino acids. As a result of a phylogenetic analysis of SDR family enzymes, the BDHs were considered to comprise a cluster in dependent from the other SDR enzymes.