Gene Cloning,Expression And Biochemical Characteristics Of Novel D-amino Acid Dehydrogenase From Proteus Mirabilis JN458

D-amino acids are widely found in microorganisms,plants and animals,and can be used in clinical medicine,pharmaceutical industry,food and cosmetic industries.In order to analyze and recognize the important role of D-amino acids,a fast and accurate detection method of D-amino acid should be established.D-amino acid dehydrogenase can catalyze the dehydrogenation of D-amino acid to generate the corresponding 2-oxo acids stereospecifically.Therefore,it has great prospects that D-amino aicd is decteted by D-amino acid dehydrogenase method.In this study,the genes ecoding D-amino acid dehydrogenase in Proteus mirabilis JN458 were cloned and expressed and its expression vectors,inducing conditions and purification conditions were optimized.At the same time,its enzymatic properties were researched.The main research results were showed as follows:?1?Two genes encoding D-amino aicd dehydrigenase in P.mirabilis JN458 were cloned successfully and were ligated with pCold?to construct a recombinant plasmid and overexpressed in Escherichia coli BL21?DE3?.The two sequences were as follows:Pmdad1?1305 bp?and Pmdad2?1128 bp?.The molecular weights calculated from the translated amino acid sequence were 47.72 kDa and 40.83 kDa,respectively.By comparison,only the enzyme encoded by the gene Pmdad1 had activity.?2?Since the expression amount and enzyme activity of PmDAD1 were not high,the expression vectors and the inducing conditions were optimized.The pET28a,pRSFDuet-1,pETDuet-1 and pColADuet-1 were selected as expression vectors to construct the recombinant plasmids pET28a-Pmdad1,pETDuet-1-Pmdad1,pRSFDuet-1-Pmdad1 and pColADuet-1-Pmdad1.The expression level and enzyme activity of p ETDuet-1-PmDAD1were the highest,which was doubled compared with pCold?-PmDAD1.The optimized conditions were as follows:the initial cell concentration OD₆₀₀ was 0.4,the inducting temperature was 20?C,the IPTG concentration was 0.4 mmol·L^-1,and the inducting time was16 h.?3?The PmDAD1 protein was purified by using Ni⁺affinity chromatography and ultracentrifugation,and its purification conditions were optimized.Since PmDAD1 is tightly bound to cell membranes,it cannot be isolated and purified by Ni⁺affinity chromatography.The optimized ultracentrifugation conditions were as follows:0.015%Triton X-100,5%glycerol,and 5?mol·L^-1 FAD.The specific activity of PmDAD1 was 3.7 U·mg^-1 after purification,which was 5.6-fold than crude enzyme.?4?Enzymology properties of D-amino acid dehydrogenase were studied.The following results were obtained:optimum activity was at 45?C and pH 8.0.PmDAD1 showed high stability in the temperature range of 20–30?C for 1 h.PmDAD1 showed a wide range of substrates of 17 kinds of D-amino acids.The D-alanine,D-asparagine,and D-phenylalanine were used to study the kinetic parameters.For D-alanine,PmDAD1 displayed the highest catalytic efficiency(k_cat/K_m=7.76 L·mmol^-1·s^-1)and maximum affinity(K_m=2.07 mmol·L^-1).