Gene Mining And Molecular Modification Of L-aspartate α-decarboxylase And Its Application In Catalytic Synthesis Of β-alanine

L-aspartateα-decarboxylase（PanD）specifically catalyzes the decarboxylation of L-aspartate to produceβ-alanine and is a key enzyme in the biosynthesis ofβ-alanine,pantothenic acid,carnosine and other compounds.Among them,β-alanine is an important building block for the synthesis of a variety of pharmaceuticals,and also has a wide range of applications in food,feed and cosmetics,and is one of the 12 most promising three-carbon compounds.Enzymatic synthesis ofβ-alanine is currently the mainstream method forβ-alanine synthesis due to its greener,more efficient and sustainable nature.PanD enzymes of Corynebacterium glutamicum and Bacillus subtilis168 origin are commonly used,but the bottleneck for the synthesis ofβ-alanine or its downstream products by these two sources is the lower enzyme activity.To solve this problem,this study carried out gene mining and molecular modification of PanD,and enhanced the catalytic performance of the new enzyme through semi-rational design to establish a whole-cell catalytic synthesis ofβ-alanine,with the following results:（1）In this study,a new L-aspartic acidα-decarboxylase was discovered,whose enzymatic properties were superior to those of existing enzymes.Combined with the available sequence information of L-aspartateα-decarboxylase（PanD）,L-aspartateα-decarboxylase from B.subtilis 168 and C.glutamicum origins,which are capable of forming acetonyl groups,were selected as probes,and six L-aspartateα-decarboxylase genes from different sources were identified by BLAST analysis based on the Uniprot protein database.Whole-cell catalytic activity comparison,pure enzyme activity and enzymatic properties were performed,respectively.L-aspartateα-decarboxylase（PanD）derived from Corynebacterium jeikeium was mined and the wild enzyme enzyme catalytic activity was higher than that of the other sources being used.The activity of PanD enzyme from C.jeikeium was 9.8 U·mg^-1,which was the highest among the 6 PanDs,and its affinity for substrate was the best,K_M value was 3.6±0.8mmol·L^-1.The catalytic efficiency of PanD enzymes derived from C.jeikeium was further improved by molecular modification.（2）In this study,the function evolution of L-aspartateα-decarboxylase derived from C.jeikeium was successfully achieved by structure-based semi-rational design,and the activity and stability of the enzyme were improved.Alpha Fold2 was used to predict the protein structure of L-aspartateα-decarboxylase derived from C.jeikeium and perform molecular docking with the substrate.Amino acid residues in the binding pocket 5（?）range were virtual mutated by sequence conservation analysis and binding free energy module in Rosetta,and non-conserved sites with reduced free energy（ΔΔG）after mutation were selected for CAST saturation mutation.After preliminary screening by whole cell catalytic system and re-screening after enzyme purification,four mutants with improved activity were obtained,namely R3K,C26V,I88M and Y90F.The enzyme activity of the combined mutant I88M-Y90F-C26V-R3K was 25.5U·mg^-1,which was 2.6 times higher than that of the wild type(9.8 U·mg^-1).Kinetic analysis showed that the K_M value of the mutant was 2.3 mmol·L^-1,which was significantly lower than that of the wild type(3.6 mmol·L^-1),indicating that the mutant had a stronger affinity for the substrate.The Tm value of the mutant was 82℃,and its stability was improved compared with that of the wild type.（3）In this study,molecular dynamics simulation（MD）was used to analyze the the three dimensional structural changes before and after the mutation.The results showed that the RMSD value of the mutant was slightly lower than that of the wild type,and the mutant had higher stability than the wild type.By analyzing the conformation of the substrate pocket before and after the enzyme mutation,it was found that isoleucine at 88 was changed to methionine,and the orientation of the terminal methyl group changed from Lys9 orientation to laterally orientation.After the mutation of tyrosine at position 90 to phenylalanine,there is no hydroxyl substitution on the benzene ring para-site,and the relative position of Gly24 and Lys9 is closer to the substrate L-aspartic acid.The distance between the amino group of Lys9 and theα-carboxylic group of L-aspartic acid is shortened from 2.8（?）to 2.2（?）,and the binding with theα-carboxylic group of L-aspartic acid is closer.It is beneficial to catalyze decarboxylation.The mutation of site 26 to valine is beneficial to the correct folding of enzyme.There is a strong dynamic coupling between site 3 and the active center,and the disturbance at site 3 will be transmitted to the active center.（4）In this study,a high-density fermentation and whole-cell catalytic process of mutants in E.coli was established.The I88M-Y90F-C26V-R3K mutant was fermented in a 5 L fermenter,and the inducer IPTG was added at an interval of two hours after the bacterial density reached OD₆₀₀=60,and the final concentration of IPTG was 0.6 mmol·L^-1.The fermentation volume reached OD₆₀₀=120 at about 26 h,and the maximum enzyme activity was 2853.87 U·m L^-1.Whole-cell catalysis with OD₆₀₀=40 resulted in the complete conversion of 1 mmol·L^-1substrate in 12 h.The yield ofβ-alanine reached 87.6 g·L^-1with a molar conversion rate of98.3%.