Asymmetric Enzymatic Deduction Of Frochiral Ketones Compounds By Enzyme-coupled System

During the process of taking the aldo-keto reductase as a catalyst to synthesize chiral compounds,coenzymes will be consumed at the same time. These coenzymes are more expensive than the prepared products. Meanwhile, the stabilities of these coenzymes are poor, and it is difficult to reuse these coenzymes. In industry production, it is less likely to cast a large amount of coenzymes. Therefore, the construction of the highly efficient cofactor regeneration system and the recycling use of coenzymes are the problems that must be solved in the present.In order to solve the problem of cofactor regeneration, gene engineering technique was used in this paper. With the genomes of Lodderomyces as template for polymerase chain reaction(PCR)amplification, formate dehydrogenase(FDH) was obtained. By connecting with the carrier pET28 a,recombinant plasmid p FDH-pET28 a was obtained. Then the plasmid was transformed into E. coli BL21(DE3) to obtain E. coli BL21-FDH. In this paper, the influences of the temperature, the concentration and the time on the soluble protein expression were studied. The enzyme activity of FDH was determined.Two kinds of pharmaceutical intermediates, L-2-aminobutyric acid and(S)-1-(2,6-Dichloro-3-fluorophenyl) ethanol, were synthesized by the highly efficient cofactor regeneration system.The plasmid p TD-pET28 a of threonine deaminaseand(TD) and pLDH-pET21 a of leucine dehydrogenase(LDH) were constructed, respectively. By transforming pTD-p ET28 a into E. coli BL21(DE3), E. coli BL21-TD was obtained. The enzyme activity of TD was determined. By transforming recombinant plasmid pFDH-pET28 and pLDH-pET21 a into E. coli BL21(DE3), E. coli BL21-FDH/LDH was obtained. The enzyme activity of LDH was determined. L-2-aminobutyric acid was synthesized by enzyme-coupled system. The conditions of conversion reaction(temperature, pH, the initial concentration of nicotinamide adenine dinucleotide(NAD+), the maximum amounts of substrate, the reaction time, etc.)were explored to obtain the optimal condition. Meanwhile, the optimal purification conditions of L-2-aminobutyric acid were optimized by controlling factors(temperature, pH, the washing times of alcohol, etc). L-2-aminobutyric acid was characterized by mass spectrum(MS), nuclear magnetic resonance(NMR) and optical rotation. The purity and yield of the product were 97% and 80.65% respectively, whichhave reached the requirements of the following experiments.The plasmid pADH-pET21 a of ethanol dehydrogenase(ADH) was constructed. By transforming recombinant plasmid pADH-pET21 a and p FDH-pET28 a into E. coli BL21(DE3), E. coli BL21-FDH/ADH was obtained. The enzyme activity of ADH was determined.(S)-1-(2,6-Dichloro-3-fluorophenyl) ethanol was synthesized by enzyme-coupled system. The conditions of conversion reaction(temperature, pH, the maximum amounts of substrate, the cosolvent, the reaction time, etc.) were explored to obtain the optimal condition. The product was purified by column chromatogram.(S)-1-(2,6-Dichloro-3-fluorophenyl) ethanol was characterized by MS, NMR and optical rotation. The purity and yield of the product were 100% and over 50% respectively, which have reached the requirements of the following experiments.The preparations of L-2-aminobutyric acid and(S)-1-(2,6-Dichloro-3-fluorophenyl) ethanol demonstrate the effectiveness of the construction of cofactor regeneration system.