Study On Directed Evolution Of L-aspartate-α-decarboxylase And The Catalytic Synthesis Of β-Alanine By Three-enzyme Cascades

β-alanine is a non-protein amino acid,widely used in chemical,pharmaceutical,material,feed and food fields,its green synthesis is gaining more and more attention.There have been many reports that the use of enzymatic synthesis ofβ-alanine has the advantages of mild reaction conditions,high selectivity,and few pollutants.Among them,the use of L-asparate-α-decarboxylase to decarboxylate L-aspartic acid to generateβ-alanine is one of the most popular methods currently studied.However,the yield ofβ-alanine synthesized by the Pan D enzyme method is not high,and there is the effect of substrate inhibition;on the other hand,the raw material cost for producingβ-alanine by L-aspartic acid is relatively high.Therefore,the development of enzyme catalysts with higher activity and stability or the development of new technological routes using more economical raw materials is an important basis for their industrial application.In this study,the directed evolution of L-asparate-α-decarboxylase（Pan D）derived from Corynebacterium glutamicum was studied to improve its enzyme activity or stability,on On the other hand,inexpensive and easily available maleic acid was constructed selected as a substrate for the synthesis ofβ-alanine,Through through a novel the pathway of by using maleic isomerase（Mai A）,L-aspartase（Asp A）and L-aspartate-α-decarboxylase（ADC/Pan D）as cascade catalytic synthesis ofβ-alaninebiocatalysts.,and study t The impact effect of various factors on the tandem reaction as well as Optimizing the reaction conditions optimization were systematically studied for efficiently and finally achieving the goal of efficient biocatalytic synthesis ofβ-alanine.In the first part of the paper,the error-prone PCR method was used to study the directed evolution of L-asparate-α-decarboxylase（Pan Dcg）derived from Corynebacterium glutamicum.The mutation library was successfully constructed by two rounds of PCR,with a mutation library capacity of 1600.The phenolphthalein color method was used for high-throughput screening.After rescreening and sequencing verification,6 six sense mutants were obtained,and protein purification and enzymatic properties were studied for these 6selected mutants.The results show that no mutants with significantly higher enzyme activity than the maternal enzyme have been found,but two mutants have been found to have higher thermal stability than the maternal enzyme.The enzyme activity of the mutant L121P（5.64 U/mg）is the same as that of the maternal enzyme（5.66 U/mg）,but the residual enzyme activity obtained after incubation at 50°C and 70°C for 12h is 7%and 17%higher than that of the maternal enzyme and 17%respectively,indicating that its thermal stability is superior to the maternal enzyme.The enzyme liquid activity of the mutant L91I&A110T（3.45 U/mg）is only 61%of the maternal enzyme activity,but it can still maintain 94%of the residual initial enzyme activity after being incubated at 50°C for 12 h,while the maternal enzyme is only 74.6%.It shows that the thermal stability of the mutant L91I&A110T is significantly quite better than the maternal enzyme.In the second part of the paper,a maleic acid substrate was selected as a cheaper substrate for the synthesis ofβ-alanine,through a three-enzyme cascade catalysis route.by used to pass through maleic cis-trans isomerase（Mai A）,L-aspartase（Asp A）and L-aspartate-α-decarboxylase（ADC/Pan D）three enzymes cascade catalytic synthesis ofβ-alanine reaction pathway.First,the co-expression strains of Mai A and Asp A were constructed,and the double-enzyme reaction conditions of the co-expression strains were preliminary explored.It was found that the double enzymes had good catalytic activity between p H 6.5 and 8.5.When the cell volume amount reached was 6 g/L,the substrate concentration of 400 m M can reach 97.5%conversion after 6 hours of reaction.Secondly,then compared the catalytic ability of three L-asparate-α-decarboxylase.Through the study of cell permeability,it was found that after 0.4%CTAB treatment of the cells,the enzyme activity of ADC cells was increased to make it The enzyme activity is about 25 times higher than that of untreated one.Therefore,the ADCtb after permeation treatment was selected for the subsequent three-enzyme cascade reaction,and the single-enzyme reaction optimization was detailed studied because the decarboxylation isperformed for the rate-limiting step decarboxylation reaction in the three-enzyme cascade reaction.The results showed that when the amount of coenzyme was 4 m M and the reaction temperature is 37°C,the buffer system is 0.1 M PBS buffer（p H=6.5）,and the amount of catalyst is 30 g/L.The ADCtb can completely convert 400 m M substrate within 6 h.Finally,on the basis of the above experiments,the three-enzyme cascade catalytic reaction was studied,The factors such as enzyme addition ratio,enzyme addition method,substrate concentration and reaction volume were studied respectively.The results show that the one-step enzyme addition method is more conducive to the tandem reaction of the three enzymes.When the co-expression strain and ADC_tb are added at a ratio of 1:5,a reaction time of 400 m M substrate concentration for 6 h can give a space-time of 136.3 g/L/d The yield is higher than 89.2 g/L/d when the enzyme is added stepwise;when the substrate concentration is increased from 400 m M to 600m M,the molar conversion rate is changed from 95.6%to 94.7%.After 6 hours of reaction,the space-time yield ofβ-alanine can reach 203 g/L/d,and the catalytic reaction efficiency is further improved;Further increase the substrate concentration to 800 m M,and it can also be completely converted after 9 h.The molar yield of the product reaches 93.9%,indicating that three-enzymes tandem reaction system can synthesizeβ-alanine at low cost and high efficiency under high substrate concentration,which has potential application value.