Enzymatic Properties,Direction Evolution And Application Of Alanine Dehydrogenase From Helicobacter Aurati

Alanine dehydrogenase(ADH;EC.1.4.1.1)belongs to redox enzymes,which can use NAD(P)⁺and NAD(P)H coenzyme to catalyze the conversion between alanine and pyruvate.Alanine dehydrogenase is ubiquitous in bacteria,fungi and plants,it is a very important enzyme in carbohydrate metabolism and amino acid metabolism.It has been widely used in the fields of food,medicine and biotechnology.In this study,the gene sequence of Helicobacter aurati was analyzed,and found it had two potential ADH sequences.We named it HaADH1 and HaADH2,the plasmids with different copy number promoted its expression with the help of molecular chaperone protein.Using pyruvate as substrate,we found that HaADH1 could catalyze the conversion between alanine and pyruvate,but HaADH2 showed no catalytic activity for both alanine and pyruvate.Based on the study of the enzymatic properties of HaADH1,it was found that the optimum temperature of reduction reaction was 55?,and the optimum p H for reduction reaction was only 17%of the maximum activity at p H 5.0,but the specific enzyme activity was 45.6 U·mg^-1,and the optimum p H for oxidation reaction was 9.0.All the measured metal ions have different degrees of inhibition.In the reduction reaction,HaADH1 showed the highest substrate affinity for pyruvate(K_m=0.6 m M,k_cat/K_m=364.1 s^-1·m M^-1).Compared to pyruvate,oxaloacetic acid2-ketobutyric acid,3-fluoropyruvate,?-ketoglutaric acids,glyoxylic acid showed a residual activity of 93.3%,8.9%,5.6%,2.6%,2.5%,respectively.Phylogenetic tree analysis showed that this was a new type of ADH which had a low sequence similarity to available ADH reported in references.HaADH1 and glucose dehydrogenase genes were ligated to p RSFDuet-1,which was transformed into E.coli for co-expression and catalyzed by whole-cell catalysis for 12 h,the conversion rate of 3-fluoropyruvate reached 98.3%for 50 m M.The four positions of Arg 15,Lys 74,His 95,Asp 268 were found to be very conservative compared to the alanine dehydrogenase of previously literature.These mutants lost their activity after site-directed mutagenesis.According to previous literature,these sites have been documented to be catalytic center,so it's likely that they're also catalytic center in HaADH1.Using the model structure obtained by homologous modeling,alanine scanning of amino acids in the range of 5(?)around the substrate revealed that only the catalytic activity of Y93A for pyruvate increased to 106%of that of the wild type(WT).Saturated mutation of three amino acids Tyr 93,Leu 128,Met 131 in the hydrophobic pocket of alanine side chain methyl showed that the activity of pyruvate reductase of Y93R and Y9K increased to 164.5%and 155.0%of that of wild type,and the half-life was increased to 3 h and 3.4 h,respectively.The k_cat/K_m of3-fluoropyruvic acid increased from 0.6 s^-1 m M^-1 to 2.5 s^-1 m M^-1 and 1.8 s^-1 m M^-1,respectively.