Engineering Of The Conformational Dynamics Of Enzymes To Improve Properties To Produce Pharmaceutical Intermediates

Biocatalysis is widely used in the production of pharmaceutical intermediates with advantages of environmental protection and high efficiency.Biological catalyst?enzyme?plays key role in the process of biocatalysis,and its activity,selectivity,stability will directly affect the catalytic efficiency and large-scale industrial application.Therefore,many protein evolution strategies have been developed to improve the characteristics of enzymes.Engineering of enzyme conformational dynamics has become an effective strategy for protein evolution,achieving significant results in terms of relieving product inhibition and for the rational design of enzymes.In this study,we established the conformational dynamics engineering method,and applied to increase the enantioselectivity of Candida antarctica lipase B?CALB,EC 3.1.1.3?for?R?-3-TBDMSO glutaric acid methyl monoester preparation and to relieve the product inhibition of L-amino acid deaminase?L-AAD,EC 1.4.3.2?for phenylpyruvic acid?PPA?preparation.Major results are listed below:1.The establishment of the engineering of conformation dynamics.By analyzing the important role of conformation in the process of catalysis,the engineering of conformation dynamics is proposed.Also,the process of the engineering of conformation dynamics was established:homologous modeling,molecular docking,selection of mutation sites,the design of best mutants by molecular dynamics simulation,evaluation of the mutant.The softwares?Swiss-model,Autodock Vina,GROMACS 4.5.5?were selected and transplanted to the supercomputer Sunway TaihuLight System and completed the construction of the simulation platform of the conformational dynamics engineering method.2.Engineering of the conformational dynamics to increase enantioselectivity.Based on structural analysis and molecular dynamics simulations,two key residues?D223 and A281?were identified,and mutant M₀?D223V/A281S?was designed to decrease the conformational dynamics of the pocket and channel.Computational evaluation clearly indicated that the decreasing conformational dynamics led to a strengthening of the structural constraints of the pocket and channel.We compared the distances between H224 and the oxygen of the R-enantiomer?distance d1?and the S-enantiomer?distance d2?,as well as the corresponding binding energies.The mutant M₀ exhibited decreases in d1,increases in d2,and larger differences in the binding energies(from 3.86 kJ?mol^-1 to 17.49 kJ?mol^-1)of the R-and S-enantiomers as compared with those of the EF5 mutant.Experimental evaluation was performed for mutants,with the mutant M0 exhibiting high R-enantioselectivity?99.00%;increased from 8.00%?.The kinetic parameters of the CALB mutants were determined,consistent with enantioselectivity.The induction and transformation conditions were optimized.The highest activity(15000 U?mL^-1)of CALB mutant M₀ was achieved when the recombinant GS115 was induced at 28^oC with glucose for 48 h.The optimum transformation system:30^oC,pH 8.0,immobilized enzyme 80 g?L^-1,substrate 60 g?L^-1.The conversion yields of?R?-3-TBDMSO glutaric acid methyl monoester were>90.00%using the CALB mutant M0 in a 3-L scale at 30°C,and the ee_R value and the space-time yield were 99.00%and 107.54 g??L?d?^-1,respectively.3.Engineering of the conformational dynamics to relieve the product inhibition.We selected mutation candidates based on the analysis of the structure and the following considerations:to avoid large alterations in main-chain dynamics and secondary structure distortion,candidates were mainly inserted within the loop regions of the pocket,excluding proline and glycine.Based on the engineering of the conformational dynamics,candidate residues were substituted with alanine.We obtained five mutants?T105A?E145A?S412A?E340A and E417A?with higher yield than wild type.The maximum yield?T105A?is 47.18g?L^-1.Combinatorial mutant was performed and mutant M₅ was achieved with the yield of 69.50g?L^-1.The mutant M₅ have significantly increased K_PI value of 84.40±2.2 g?L^-1 from 22.80±0.9 g?L^-1.The turnover numbers(k_cat)of the mutant M₅ increased by about 2-fold in comparison to those of the wild type.The catalytic efficiencies(k_cat/K_M)of the mutant M₅ remained comparable to that of the wild type.The preparation of PPA with mutant M₅.The optimised induction medium was TB.The highest activity?50 U?of L-AAD mutant M₅ was achieved when the recombinant E.coli BL21 was induced at 25^oC with 5 g?L^-1 lactose for 12 h.The reaction was catalyzed at 30^oC,pH 7.5,whole-cell biocatalysts 30 g?L^-1,substrate 65 g?L^-1.The highest PPA production concentration reached 72.50 g?L^-1,with a conversion rate of 96.67%.