Gene Mining And Molecular Modification Of L-aspartate β-decarboxylase

L-aspartateβ-decarboxylase（Asd）,which is used for industrial production of L-alanine,catalyzes the removal ofβ-carboxyl group from L-aspartic acid to form L-alanine.L-alanine has a wide range of applications in the fields of food,daily chemical industry and medicine.Production of L-alanine by enzyme catalysis has the advantages of fewer by-products,strong stereoselectivity,and high catalytic efficiency.In this study,after screening of strains from soil,excavating highly viable Asd,performing enzymatic characterization and molecular modification,the Asds were coupled with L-aspartic acid lyase to construct a recombinant E.coli with double enzyme co-expression.We established a process for whole-cell catalyzed synthesis of L-alanine from fumaric acid.The main findings are as follows:（1）L-aspartateβ-decarboxylase（ArAsd）from Acinetobacter radioresistens was successfully sieved,then heterologously expressed in E.coli and the enzymatic properties were characterized.A strain with Asd activity was obtained by screening in amino-acids-rich soil.The strain was identified as Acinetobacter radioresistens.The ArAsd gene was cloned and expressed in E.coli BL21（DE3）.A His tag was added to the N-terminus of the amino acid sequence of ArAsd protein,and a nickel column was used to purify the target protein.The molecular weight of ArAsd protein was determined by gel filtration chromatography and the results showed that ArAsd was a homododecane.The analysis of enzymatic properties showed that the optimal temperature of ArAsd was 50oC and the optimal pH was 4.5.It was stable under the conditions of pH=6.0～7.0,and remained 30%of enzyme activity after 12hours of treatment at pH=4.5.The specific enzyme activity of ArAsd when using L-aspartic acid as substrate is 753 U·mg^-1,which is the highest specific enzyme activity reported so far..（2）Based on structural analysis,the acid stability of ArAsd was improved through molecular modification.Homology modeling and surface amino acid prediction of ArAsd were used to construct a series of mutants,among which N35D and A179E mutants showed better acid stability.The optimal pH of N35D was 6.0,and remained 82%of the enzyme activity after 12 hours of treatment at pH=4.5;the optimal pH of A179E was 5.5,and remained 87%of the enzyme activity after 12 hours of treatment at p H=4.5.Both mutants had significantly improved acid resistance.The whole-cell catalysis results showed that when the conversion time was 12 h,mutant A179E catalyzed the substrate to produce L-alanine 943.53mmol·L-1,and the molar conversion rate was 94.4%;mutant N35D catalyzed the substrate to produce L-alanine Acid 998.95 mmol·L-1,molar conversion 99.9%.（3）ArAsd and EcAspA double enzyme coupled recombinant strain were successfully constructed.ArAsd-N35D and EcAspA were co-expressed in E.coli BL21（DE3）using a single plasmid with dual promoter.The results showed that the recombinant strain BL21（DE3）/pRSF_Duet-1-EcAsp A-ArAsd-N35D has the highest substrate catalytic conversion efficiency at pH=6.0.The whole cell catalytic ability test was carried out.Under the conditions of pH=6.0,37oC and 200 r·min^-1,the whole cell catalyzed fumaric acid(1 mol·L^-1)to product L-alanine at 971.26 mmol·L^-1,and the conversion rate was 97.3%.