Lipase Coevolution And Its Catalytic Synthesis Of Chiral Drug Intermediates

Coevolution refers to the synergistic changes that occur between paired biological individuals/biomolecules and are related to the maintenance or optimization of the functions of these organisms/biomolecules.Coevolutionary analysis of amino acid levels can identify groups of functional amino acids that influence protein function and provide new ideas for the rational design of new functional proteins.However,the existing method of protein coevolutionary analysis is fully dependent on sequence information,which would cause low accuracy in high-throughput calculation,and the lack of effective experimental verification methods makes it difficult to provide specific ideas for the molecular evolution of enzymes.Therefore,developing the method of protein evolution guided by coevolutionary network is of great significance.In this study,the co-evolution of Candida antarctic lipase B(CALB)at the family level was studied.Based on the covariant residue network,the function of CALB was modified,and the CALB mutant was used to synthesize chiral drug intermediates:1.Enzymatic function identification of CALB covariant residues:In the previous laboratory work,we set up a new coevolution analysis method,SCA.SIM,by integrating structure information and MD simulation data with SCA.Here,the 38predicted covariant residues are tested for function by high-throughput quantitative evaluation in combination with activity and thermostability assays of a mutant library and deep sequencing.In general,most mutations show significant sensitivity to activity or thermostability,which indicates that covariant positions are functionally important.As control,we randomly selected 33 non-covariant sites(about 10%residues of CALB)and mutated them to cysteine.The results show these 14 mutants have little effects on bioactivity.This further supports that the predicted covariant sites with SCA.SIM have important functions.2.Enzyme directed evolution guided by co-evolution:Based on the landscapes of both activity and thermostability,most mutants play key roles in catalysis,and some mutants gain 2.4-to 6-fold increase in half-life at 50°C and 9-to 12-fold improvement in catalytic efficiency.In order to further obtain better mutants,we tried the iterative saturation mutation technique(ISM)and the two-point saturation mutation technique to combine these sites.We created a saturated mutant library(?3000 colonies)for a pair sites of A225 and T103.The best mutant A225F/T103A increased the activity about 32-fold than wild type CALB.Dynamic simulation experiment shows that the mutation A225M induced significant conformation change at the opposite?-helix,which forms more hydrophobic interactions between enzyme and substrate.This can partly explain A225M with high catalytic efficiency.We explored the interaction between the mutant site and other amino acids and the overall interaction within the protein,and found that the increased stability of the mutant may be due to the increased overall interaction within the protein.Therefore,we can suppose that these sites might play some key roles in protein activity and thermostability based on the complexity of interactions between amino acid residues.The SCA.SIM algorithm can be used for enzyme engineering research.3.CALB lipase mutants catalyze synthesis chiral 2-substituted 2,3-dihydro-1,4 benzodioxane motifs:chiral 2-substituted 2,3-dihydro-1,4 benzodioxane motifs have wide application in a variety of bioactive natural products and therapeutic agents with important biological activities,such as the therapeutic agents piperoxan,prosympal,dibozane,and doxazosin.Using 1,4-Benzodioxane-2-carboxylic acid methyl ester as the substrate,after screening the CALB mutants obtained before,we found that mutant A225F and A225F/T103A can catalyze kinetic resolution of the substrates.The influence of the co-solvents and its concertration upon kinetic resolution demonstrated that in the presence of 20%n-butanol as a co-solvent,resulted in the optimal resolution of the substrate(ee_s 97%,E=278)at 50 m M substrate concentration.In this paper,we developed an enzyme engineering method based on the coevolution network to guide CALB evolution,verified the reliability of coevolution analysis algorithm SCA.SIM.And CALB mutant was successfully used to synthesize chiral 2-substituted 2,3-dihydro-1,4 benzodioxane motifs.The results indicate that this study might have great potential for the coevolution analysis of proteins and useful tool for the directed evolution design of enzymes.