One-pot Enzymatic Synthesis of L-2-Aminobutyric Acid Coupling with a NADH Regeneration System Based on Ketoreductase
LIU Shan;LI Yuan;ZHU Jun;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences;
A process of one-pot enzymatic synthesis of L-2-aminobutyric acid was studied. The ilva gene encoding L-threonine deaminase( L-TD) from E. coli,leudh gene encoding leucine dehydrogenase( Leu DH)from Geobacillus stearothermophilus and kred gene encoding ketoreductase( KRED) from Novosphingobium aromaticivorans were cloned into p ET-21a( +),then introduced into E. coli BL21( DE3) respectively. All of the three enzymes were overexpressed in soluble forms. L-2-aminobutyric acid was efficiently synthesized with low-cost L-threonine as substrate by the recombinant L-TD and Leu DH coupling with a NADH regeneration system based on KRED. Effects of p H and the concentration of L-threonine and isopropanol on the yield of L-2-aminobutyric acid were investigated. After 20 h at the optimal p H 7. 5 ~ 8. 0,the yield of L-2-aminobutyric acid could achieve 99%,with a concentration of 43 g / L,when using 50 g / L L-threonine,5% isopropanol,0. 5g / L NAD~+,0. 6g /L L-TD,2g /L Leu DH and 2g /L KRED. A new thought for the production of L-2-aminobutyric acid was provided.