Study On Enzymatic Preparation Of D-Histidine And D-Tryptophan

D-histidine and D-tryptophan are important heterocyclic amino acids,and are widely distributed over microorganisms?plants and animals.D-histidine and D-tryptophan had important application in pharmaceutical?food and chemical industries,D-histidine is important resolution agent and precursor of peptide drug,and D-tryptophan is the synthetic ingredients of Octreotide,Calais and other drugs.D-tryptophan can be prepared by chemical synthesis,enzymatic synthesis,chiral resolution of the racemate and fermentation,etc.The method of D-histidine preparation is mainly chemical asymmetric transformation.Compared with chemical synthesis or chemical chiral resolution,biosynthesis or biological chiral resolution can yield D-AAs with high optical purity,green process.Therefore,D-histidine and D-tryptophan were prepared by enzymatic method in this study.The main work and results are as follows:1.A two-enzyme cascade system was developed to produce D-histidine.In this study,the fusion expression strategy was used to optimize the expression of AAR and HDC in the prokaryotic system,and the recombinant pGEX-KG-AAR/BL21(DE3)and recombinant pSUMO-HDC/BL21(DE3)were obtained with high enzyme activity.First,L-histidine was racemized to DL-his by Amino Acid Racemase(AAR),then the L-histidine in raceme were selectively degraded to histamine by Histidine Decarboxylase(HDC),so that D-histidine could be easily extracted from reaction system.The enzymatic characteristic of the two enzymes were investigated,and the reaction system of D-histidine was prepared by the two-enzyme cascade method.Finally,800 mmol/L of L-histidine was used as the starting substrate for the preparation of D-histidine.The yield of D-histidine was 46.3%and the optical purity was 98.2%.2.Study on the Resolution of DL-tryptophan by L-Aromatic Amino Acid Decarboxylase.The Bacillus atrophaeus C89 A ADC gene was cloned into the prokaryotic expression system to express L-Aromatic Amino Acid Decarboxylase(AADC).AADC was used to split DL-traptophan because of selective degradation of L-tryptophan.The enzyme reaction conditions and separation system were optimized.Finally,20 g/L DL-tryptophan was used to get D-tryptophan,the yield of D-tryptophan was 42.5%,and the optical purity was more than 99%.