RESUMO
The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.
Assuntos
Esterases/genética , Genes Bacterianos/genética , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Meios de Cultura , Enzimas de Restrição do DNA , DNA Bacteriano/análise , Escherichia coli/enzimologia , Esterases/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Pseudomonas/genética , Recombinação GenéticaRESUMO
The esterase catalyzes the stereoselective hydrolysis of methyl DL-beta-acetylthioisobutyrate (DL-ester) to give D-beta-acetylthioisobutyric acid (DAT). To use the enzyme for DAT production, the esterase gene of Pseudomonas putida was cloned and expressed in E. coli. The recombinant E. coli containing the esterase gene produced a large amount of the enzyme in an active form. This strain could be used for the asymmetric synthesis of DAT.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Butiratos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Esterases/genética , Esterases/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Clonagem Molecular , Esterases/biossíntese , Expressão GênicaRESUMO
Methyl L-ß-acetylthioisobutyrate was racemized with 1,8-diazabicyclo-[5.4.0]-undecene-7 as a catalyst. Methyl methacrylate and thioacetic acid were identified as the intermediates of the reaction. Thioacetic acid was relatively unstable and susceptible to decomposition during the racemization process. The addition of excess methyl mechacrylate to the reaction mixture prevented a decrease of the racemate. The racemized ester was confirmed to be usable as a substrate for the enzymatic production of D-ß-acetylthioisobutyric acid.
