Molecular Modification And Enzymology Properties Of Leucine Dehydrogenase

Leucine dehydrogenase?LDH?is a NAD⁺dependent oxidoreductase which reversibly catalyzes the hydrolysze of L-leucine and other branched-chain L-amino acids to the corresponding keto acids and their analogs.The leucine dehydrogenase in this thesis is used to hydrolyze of butanone acid to L-2-aminobutyric acid.L-2-aminobutyric acid is a very important pharmaceutical intermediate which is widely used in the synthesis of antituberculotic ethambutol,brivaracetam,and anti-epileptic levetiracetam.The leucine dehydrogenase used in this project is a recombined enzyme which is previously generated in our laboratory.In order to screening mutant strain faster we establish a high-throughput screening model based on calcein-copper first,then optimized its parameters.The results showed that the optimum substrate system was 1 ml reaction system contains 0.01g butanone acid,0.07 g NADH,10?L ammonia.The calcein concentration of the system is 1.0×10^-5 mol/L;the copper ion concentration of the system is 1.5×10^-4 mol/L,the optimized reaction pH is 7.5,the stability of the reaction time is 20 minutes,the optimized excitation wavelength and emission wavelength,490 nm and 520 nm,respectively.The product standard curve measured by this method is good in linearity,and the results in agreement with the measured data of amino acid analyzer.We analyze the properties of leucine dehydrogenase through expasy and npsa-prabi online website.Then we know the molecular weight of we leucine dehydrogenase is 40571.3Da.It is composed of 367 amino acid residues,calculated theoretical isoelectric point is 5.56,instability index?II?value is 32.01,fat index is 84.55,the hydrophilic average coefficient is 0.304,and the alpha helix in the secondary structure is more.Use the Swiss-online-Model software to build the homologous modeling that gets three Models.Using the evaluate software-services to evaluate three models respectively.Then we use model 1?the best?docking the butanone acid molecular to get the reasonable conformation.The reasonable conformation is analysis on PYMOL and combining mechanism reported in the literature research,we selecting Leu41,Gly43,Arg61,Met66,Asn70,Asn262,Met347,and Gln358 perform the saturation mutagenesis.Finally the muatnts with the mutations of M347G,M347N,Q358T,Q358N,N70F,M347G/Q358T has showed higher enzyme activity.The three mutant strains M347G,Q358T,M347G/Q358T and the original strain were selected to analyze the enzymology properties.Specific enzyme activity in mutant M347G,Q358T and M347G/Q358T is3.41,4.59 and 4 times than the original strain,respectively.Enzymatic reaction kinetics results showed that the K_m value of mutant is lower than the original strains and the k_cat and k_cat/K_mvalue of Mutant is higher than the original strains.The optimal reaction temperature of original strain is35? and the mutant M347G/Q358T at 30?,35? and 40? has the same enzyme activity.The thermal-stability of the mutant temperature is better than the original strain,and?best?stability is under the condition of 40?.The optimum pH of reaction is 9.0,and eucine dehydrogenase showed better stability in the weak alkaline environment.Finally,the influence of metal ions and surfactant in the reaction of leucine dehydrogenase before and after the mutation can be neglected.