Study On Synthesizing Chiral Amino Acid By Engineering Leucine Dehydrogenase With Structural Model Analysis

Leucine dehydrogenase(LeuDH,EC 1.4.1.9)is a key enzyme which has a widespread sources and is widely involved in the metabolism and synthesis of many branched-chain amino acids in the body.It can reversibly catalyze interconversion between L-amino acids and corresponding α-keto acids under the action of the cofactor NAD(H).Due to its preference for NAD(H)cofactors and limited substrate spectrum,its application is limited in industrial catalysis.D-amino acids in unnatural amino acids(UAAs)also play an important role in the field of medicine.At present,it has reported that only membrane-bound protein meso-2,6-diaminopimelate dehydrogenase(mesoDAPDH)can catalyze ketoacids to produce corresponding D-amino acids,but its catalytic activity is very low.It is necessary to change the substrate profile of LeuDH through directed evolution for the study of D-amino acid biosynthesis.At present,protein engineering is a powerful method to obtain the dominant enzymes by modifying the structure of enzymes.However,there are two problems in the application of directed evolution technology,which are the construction of effective gene mutation library of covering enzyme protein and the high-throughput screening of target protein.In this paper,we have established an efficient SDASM semi-rational design method to engineer EsiLeuDH from Exiguobacterium sibiricum 255-15.We hope to obtain a mutant protein which can change the limited substrate spectrum of EsiLeuDH and synthesize high-value chiral amino acids including D-amino acids.The main research contents of this paper are as follows:1.The recombinant strain BL21(pET28-EsiLeuDH)was constructed,which could exp ress EsiLeuDH protein efficiently.The results showed that EsiLeuDH had activity to eight linear chain L-amino acids,but no activity to D-amino acids.The enzymatic properties of EsiLeuDH were studied,it was found that the optimal pH and temperature were 9.5 and 50℃,respectively.In addition,the effect of metal ions on the enzyme activity of esileudh was determined.it was found that EsiLeuDH was completely inactivated in the presence of Ag+because of its-SH group.The stability of the enzyme was evaluated by measuring the half-life at different temperatures.The half-life of EsiLeuDH at 30℃ was up to 300 h,which showed good stability.The catalytic mechanism of natural substrate L-Leu catalyzed by EsiLeuDH was analyzed by measuring the kinetic parameters of EsiLeuDH,combining with fluorescence spectrum,UV spectrum and molecular docking methods.2.An efficient semi-rational design method of SDASM based on the protein structure model of esileudh was established.Through this method,the active site of EsiLeuDH was designed,and the amino acid residues of K89 and A122 were precisely locked to construct a small intelligent mutation library.After screening,the mutant strains K89T and A122G was obtained.The two mutant proteins were expressed and purified,and their substrate profiles were determined.The results showed that single mutant K89T had catalytic activity for Asn and Thr,and A122G had catalytic activity on Phe,His and Met,but WT EsiLeuDH has no catalytic activity on the above amino acids.Furthermore,the catalytic activity of A122G for L-Ile was as high as 5500 mU/mg.The results also showed that the optimum reaction temperature of K89T and A122G were 35℃ and 55℃,and the optimum reaction pH was 11.5 and 10,respectively.In addition,the catalytic mechanism of the single mutant binding to the substrate was analyzed by combining the kinetic and structural interaction diagrams.3.In order to further verify the effectiveness of SDASM method,we applied SDASM semi-rational design method to engineer EsiLeuDH to change the chiral selectivity for the synthesis of D-amino acid.The selected G51 and L52 sites of the EsiLeuDH were mutated by site directed saturation,respectivity.The mutant G51K may had the ability to catalyze the synthesis of D-tert-Leu.The activity of mutant L52C to 10 amino acids including L-Val,Leu,Ile and Met was higher than that of WT.On this basis,we constructed a double mutant G51K/L52C,which had the activity of LVal,Leu,Ile,Met,Ala and Lys.The highest enzyme activity of L-Ile was 6.5 U/mg,similar to WT,but the activity of L-Val and Leu is much lower than WT.In addition,the optimum reaction temperatures for the oxidative deamination reaction of G51K,L52C and G51K/L52C mutants was 45℃,35℃ and 45℃,and the optimum reaction pH was 11.5,10.5 and 10.5,respectively.In addition,the kinetic parameters of each mutant were determined and the mechanism of their binding to the substrate were analyzed.