Study On The Production Of D-amino Acids Bio-catalyzed By Double Enzymes Cascade

D-amino acids(D-AAs)are natural amino acid and exists widely in microorganisms,plants and animals.For the special biological properties,D-AAs widespread apply in medicine,food additives and other various industries,D-AA can be prepared by chiral resolution of the racemate,chemical synthesis,enzymatic synthesis,and fermentation,et al.Compared with chemical synthesis or chemical chiral resolution,biosynthesis or biological chiral resolution can yield D-AAs with high optical purity,green process,and are ideal for the industrial manufacturing of D-AAs and the requirement of modern industry sustainable development.Then it has wide industrial application prospect.In this paper,the method to prepare a series of D-AAs is double enzymes cascade biocatalysis.This method can prepare most D-AAs covering the acidic,alkaline and neutral amino acids,including D-aspartic acid,D-lysine,D-allothreonine and D-tryptophan.This paper’s work is as follows:1.Using molecular cloning technology,we constructed the two strains of genetic engineering bacterium expressing the Amino Acid Racemase(AAR)in two prokaryotic expression systems pETDuet-1 and pGEX-KG respectively.These two engineering strains respectively are pETDuet-l-AAR/BL21(DE3)and pGEX-KG-AAR/BL21(DE3),hereinafter referred to as D-AAR and K-AAR.AAR can catalyze L-amino acids to DL-amino acids.By studying and comparing the enzymatic properties of D-AAR and K-AAR,the best condition of AAR were determined:transformation temperature 40℃,pH 7.0,4 mM PLP,and AAR has a low substrate specificity.At the same time,by comparing D-AAR and K-AAR,the enzymatic activity of K-AAR higher than D-AAR under the optimum condition.Through optimizing the fermentation of K-AAR,it can determine the fermentation temperature 30℃,pH 7.5,rotation speed 200 rpm,fluid volume 20 mL/100 mL,and the shake flask fermentation time 14 h in favour of bacteria growth and protein expression.Meanwhile using the 5 L fermenter to explore the K-AAR industrial fermentation process,we determine the age of inoculation fermentation strains is 10 h and the fermentation time is 15 h to obtain high quality and high yield enzyme preparation.2.This study indicates the design and investigation of two continuous reaction processes for efficient production of D-aspartic aicd including an enzymatic racemization step and an enzymatic decarboxylation step.Firstly,L-aspartic acid was racemized to the DL-aspartic acid by whole cells containing K-AAR;then eliminating AAR and adding whole cells containing aspartic acid-β-decarboxylase(Asd),L-aspartic acid in the mixture was selectively decarboxylated to L-alanine and CO2;finally D-aspartic acid was separated from mixture by isoelectric point crystallization.The yield of D-aspartic acid could reach 47%,theoretical yield of 50%,e.e.=97%.3.This study indicates the design and investigation of two continuous reaction processes for efficient production of D-lysine including an enzymatic racemization step and an enzymatic decarboxylation step.Firstly,L-lysine was racemized to the DL-lysine by whole cells containing K-AAR;then eliminating AAR and adding whole cells containing lysine decarboxylase(LDC),L-lysine in the mixture was selectively decarboxylated to 1,5-pentanediamine and CO2;finally D-lysine was separated from mixture by isoelectric point crystallization and ion exchange resin.The yield of D-aspartic acid could reach 42%,theoretical yield of 50%,e.e.=98%.4.This study indicates the design and investigation of two continuous reaction processes for efficient production of D-allothreonine including an enzymatic racemization step and an enzymatic deaminition step.Firstly,L-threonine was racemized to the racemic mixture of L-threonine and D-allothreonine by whole cells containing K-AAR;then eliminating AAR and adding whole cells containing threonine dehydratase(TDA),L-threonine in the mixture was selectively deaminized to a-ketobutyrate and NH4;finally D-allothronine was separated from mixture by isoelectric point crystallization.The yield of D-aspartic acid could reach 45.5%,theoretical yield of 50%,e.e.=94%.5.This study indicates the design and investigation of two continuous reaction processes for efficient production of D-tryptophan including an enzymatic racemization step and an enzymatic degradation step.Firstly,L-tryptophan was racemized to the DL-tryptophan by whole cells containing K-AAR;then eliminating AAR and adding whole cells containing tryptophanase,L-tryptophan in the mixture was selectively degraded to indole,pyruvic acid and NH4;finally D-lysine was separated from mixture by isoelectric point crystallization and ion exchange resin.The yield of D-aspartic acid could reach 42%,theoretical yield of 50%,e.e.=98%.The synthetic route of D-amino acids bio-catalyzed by double enzymes cascade was based on the enzymatic resolution of the racemate.In this route,L-amino acid was regarded as the starting material.And the cost of racemization by amino acid racemase was far less than chemical racemization.In addition,enzymes used in the enzymatic resolution had selectivity and specificity.So the double enzyme cascade method simplified the biosynthesis preparation process of D-amino acid,and provided a new method to prepare D-amino acid.