|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2003 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2002 : ¥2,500,000 (Direct Cost : ¥2,500,000)
The research contents : It was clarified that meso-2,3-butanediol dehydrogenase (BDH) and L-BDH are short chain dehydrogenase / reductase (SDR) enzymes and have a similar structure. Then, using the structural knowledge of these enzymes, the elucidation of molecule evolution, with respect to enzyme functions such as stereospecificity and stability, was attempted. Various BDH-mutants were prepared by exchanging the domains and the amino acids in the active sites between meso-BDH and L-BDH. Next, crystal structure analyses of some of the mutants were performed. In additions, the influence on the stereo-structure of various mutations was analyzed by molecule calculation techniques. Furthermore, the influences of the enzyme structure on kinetic parameters were analyzed. Based on the results, the molecule evolution between L-BDH and meso-BDH was considered from the standpoint of an enzyme function.
Result : Interesting results were obtained mainly on following: 1)Amino acid residues related t
o Km value, 2)Amino acid residues related to Stereospecificity, 3)Analysis of the recognition mechanism for long-chain substrates, 4)Analysis of the dehydrogenase reaction mechanism in 3R-configulation of substrate, 5)Analysis of the substrate recognition mechanism from the competitive-inhibition constant of 2-mercaptoethanol, 6)Analysis of the influence of the mutation-point on Vmax, and 7)Analysis of the structure contributing to stability. Using the elucidated information, the characteristics of the enzyme have been improved regarding structure. That is, unstable wild-type L-BDH was successfully changed to a new L-BDH with high stability. Moreover, these results offered a fundamental indicator for the appropriate alteration to other SDR enzymes having a structure similar to that of BDH.
The production and manufacture methods of L-BD were developed using the L-BDH with newly created high stability. The efficient production of L-BD was not established until now. That is, production on the thousands of mg/l level was attained at about an 80% conversion rate from diacetyl as substrate by expressing the meso-BDH and the mutant-L-BDH simultaneously in transgenic E. coli.
At this time, the above results are under contribution as some reports. Less