Study On Enzymatic Biosynthesis Of Aromatic Amino Acid

L-tyrosine and D-phenylalanine belong to the aromatic amino acid.L-tyrosine is an essential amino acid,which plays important roles in the several kinds of fields such as food,medicine and cosmetics.As intermediates of many drugs,D-phenylalanine is an important chiral compound,also involved in the systhesis of gramicidin,cyclic peptide antibiotics.The research focuses mainly on the biological synthesis of L-tyrosine and D-phenylalanine.1.In this study,the ammonia of L-aspartic acid was transferred to the phenylpyruvic acid,and L-phenylalanine with intermediate product pyruvic acid were synthesized by aspartate aminotransferase(AspAT)whole cell.Then,L-tyrosine was enzymatic synthesized when substrate phenol and tyrosine phenol-lyase(TPL)whole cell were added into the reaction system.Some factors affecting dual enzymatic catalysis were investigated.The optimal conditions were 40oC,pH 8.5,25 g/L of phenylpyruvic acid(PPA),n(PPA):n(L-Asp)=1:1.2,m(AspAT):m(TPL)=1:1,4 mmol/L of PLP,0.1 g/L of Tween 80.30 g/L of ammonium chloride had a promoting effect on dual enzyme coupled catalysis reaction.The method biological synthesis of L-tyrosine with coupled dual enzyme in dual host,not only makes full use of the intermediate product of the reaction pyruvic acid for higher value-added products,but has reference significance for the reasonable utilization of resources and green synthesis process.2.the AspAT-encoded aspartate aminotransferase(AspAT)and the TPL-encoded tyrosine phenol-lyase(TPL)are key enzymes,which were co-expressed in Escherichia coli BL21(DE3).By constructing the recombinant plasmids,we prepare L-Tyr using the method biological synthesis.Therefore,only one enzyme was injected into the reaction system,and the experimental operation was simplified.Some factors affecting the co-expressed enzymatic catalysis were investigated.The optimal conditions of recombinant system were identical with those of dual-enzymatic reaction.As compared to dual enzyme system after 12 h,we achieved the production of L-Tyr only 6.7 g/L,which was much lower,frankly.3.In this paper,the construction of co-expressed the lactate oxidase(LOD)and TPL vector,which offered a different pathway to the formation of pyruvate,is able to prepare L-Tyr.Some factors affecting the co-expressed enzymatic catalysis were investigated.The optimal conditions were 45?,pH 9.0,10 g/L of L-lactate,n(Phenol):n(L-lactate)=1.2:1;To reduce the substrate inhibition,co-expressed of both recombinant enzymes was ordered to be added in different times and multiple ways.By the way,the yield of 5.2 g/L,was achieved for L-Tyr.This method extends the source of the pyruvic acid,making it an useful exploration for the industrialization of L-Tyr enzymatic synthesis.4.The gene(PAL)encoding L-plenylalanine,from Rhodotorula glutinis,was obtained by nucleotide synthesis after rare codon optimization.The synthetic gene was digested with Nco I and EcoR I,and the isolated DNA fragments were recovered and inserted into pETDuet-1 vector.The recombinant plasmid was transformed into E.coli BL21(DE3).The reaction was optimal at pH 9.0 and 45?.0.2 g/L of Tween 80 could promote PAL activity.And also Mg2+?Ca2+had a promoting effect on PAL activity.At the beginning,40 g/L L-plenylalanine was catalyzed by amino acid racemization enzyme(AAR)into DL-plenylalanine,then with PAL,L-plenylalanine conversion rate reached 86%,finally.