Thermostability Modification Of Maleate Cis-trans Isomerase And Biosynthesis Of L-aspartic Acid

As a staple amino acid products,L-aspartic acid has been widely used in food,medicine,and chemical industries.At present,the production of L-aspartic acid mainly relies on chemical synthesis.But the shortcomings are clearly:such as serious environmental pollution,harsh production conditions,low purity of products and so on.On the contrary,enzyme-based biocatalysis overcomes these issues,and it is becoming an important way to produce L-aspartic acid.And whole-cell biocatalysis with dual-enzyme coupling doesn?t have to purify the enzyme and the intermediate products,it is also benefical to the recycling of cells,so it can greatly reduce the costs.In this study,maleate cis-trans isomerase and L-aspartic acid amino lyase were co-expressed in E.coli.Then the recombinant E.coli was used to biocatalyze maleic acid into L-aspartic acid.The main contents of this study were summarized as followed:?1?Six different MaiA and AspA co-expression systems have been built in E.coli BL21?DE3??fum AC in this study.By comparing their co-expression results and catalytic efficiency,we got the optimal co-expression strain pMA2(pRSF_Duet-1-maiA-aspA).Whole-cell biocatalysis was performed with pMA2,and the maleic acid with different concentration(1.6,2.4,3.2mol·L^-1)could be quickly?120,250,390 min?transformed into L-aspartic acid under pH 8.0,37?,200 r·min^-1 and 20%free cell(OD₆₀₀=40)plus 80%substrate conditions.The intermediate fumaric acid had almost no accumulation overall the reaction progress,and the conversion rate all above 98%.Then the RBS of MaiA pMA2 was replaced by another RBS with higher initial translation efficiency,the activity of Mai A increased 40%with the optimal strain pMA2-RBS4.And 1.6 mol·L^-1 maleic acid was completely transformed into L-aspartic acid in 80 minute using pMA2-RBS4,the conversion rate was 99.4%,no fumaric acid accumulated also.?2?Molecular modification was performed with MaiA to improve the thermostability and activity.The results revealed that G27A-K104R and G27A-G171A mutants all had greater specific activity and half-life at 55?,they were 67.6 U·mg^-1,475.7 min and 88 U·mg^-1,392.5min respectively.Then the mutants of MaiA replaced the wild-life MaiA of pMA2-RBS4,and they were used to biocatalyze 1.6 mol·L^-1 maleic acid,the maleic acid was completely transformed into L-aspartic acid in 60 minute and 40 minute by pMA2-RBS4-G27A-K104R and pMA2-RBS4-G27A-G171A,the conversion rate was 99.4%and 99.6%.Cell immobilization was performed following,by comparing diferent cell immobilization methods,the optimal cell immobilization method was Co-immobilized with Sodium alginate–Polyvinyl alcohol–Activated carbon.After 8 recycling of the immobilized cells,the relative enzyme activity was still 81%.?3?High cell-density cultivation using pMA2-RBS4-G27A-G171A was applied in 5 L bioreactor.After the optimization of fermentation conditions,the optimal conditions were as follows:The cell was cultured at 37?,the inducer IPTG was added to fermentation broth when the cell-density reached OD₆₀₀=120,and the final concentration of IPTG was 0.8mmol·L^-1,and the temperature was reduced to 30?at the same time.After 16 hours,the maximum cell activity was about 341.3 U·mL^-1.