Preparation And Application Of Amino Acid Dehydrogenase&Alcohol Dehydrogenase

This paper explored the efficient preparation of glutamate dehydrogenase P_pGluDH and alcohol dehydrogenase TbADH,which was used for coenzyme regeneration and its application in the preparation of efficient amino acid pesticide L-Phosphinothricin by double bacteria and double enzyme,and further constructed the enzyme library containing different types of amino acid dehydrogenases,andpreliminarily explored its application in the synthesis of L-amino acids.First,the enzyme production process of glutamate dehydrogenase P_pGluDH and alcohol dehydrogenase TbADH was optimized,so as to provide references for its industrial application.The fermentation medium composition of the recombinant strain E.coli BL21(DE3)/ pET-28a(+)-P_pGluDH was optimized by the combination of singlefactor method and response surface method.The enzyme activity of the unit volume fermentation liquid was increased from 49.8 U/mL to 129.1 U/mL.and then the induction conditions of the PpGluDH recombinant bacteria were optimized,and the enzymeactivity was further improved to 159.2 U/mL,which was 3.2 times compared with LB medium,we also investigated the effect of different induction conditionson the activity of TbADH enzyme,and finally the enzyme activity reached12.5 U/mL,which was 10.4 times of the LB medium.The conversion of 2-carbonyl-4-(hydroxymethyl phosphonyl)butyric acid(PPO)to L-Phosphinothricin by double enzyme coupling catalyzed by glutamate dehydrogenase PpGluDH and alcohol dehydrogenase TbADH was studied.The effects oftemperature,pH,enzyme quantity,double enzyme ratio,NH4~+ concentration and NADP~+ concentration on the catalytic efficiency of double enzyme double enzyme system were investigated in combination with the enzymatic properties of P_pGluDH.The results showed that the optimum reaction temperature was 45 C and the optimumpH was 7.5.When the substrate concentration is 0.2 mol/L,the optimum ratio of double enzyme is 1:1(P_pGluDH 10 U/mL,TbADH 10 U/mL),NH4~+concentration is 0.2 mol/L,the molar ratio of auxiliary substrate isopropanol tosubstrate PPO is 1.25:1,and the NADP~+ concentration is 0.3 mmol/L.Under these conditions,the substrate of 0.2 mol/L,can reach 100% conversion for 3 h,and the enantiomeric excess value(e.e.)of the product is more than 99%,and the optical purity is high.The space-time yield reached 289.6 g · L~-1 · d~-1.Finally,the enzyme library of amino acid dehydrogenases was constructed based on the genome mining technology,and the application of the amino acid dehydrogenase library in the preparation of L-amino acids was preliminarily explored.The enzyme library consists of 23 amino acid dehydrogenases,including 11 leucine dehydrogenases,10 alanine dehydrogenases,1 glutamate dehydrogenase and 1phenylalanine dehydrogenase.The catalytic activity of these amino acid dehydrogenases to the three valuable keto acid substrates of 2-carbonyl-4-(PPO),2-ketobutyric acid and 2-keto-3-butenoic acid was determined.It was found that a kind of leucine dehydrogenase-EacLeuDH1,which origined from Exiguobacterium acetylicum,had a high catalytic activity.It was higher than that reported in the literature,which was 1.24 times for BceLeuDH.It has potential application in the preparation of L-2-aminobutyric acid.