Engineering Thermostability Of D-carbamoylase And Mechanism Study Based On Crystal Structure

D-Amino acids can be used for the synthesis of pharmaceutical intermediates,amino acid pesticides and other high value-added products,which have important industrial application.Hydantoinase process can be used for synthesizing various D-amino acids on industrial scale,which has the advantages of green and environmental friendlyness,high stereoselectivity and economy.However,the D-carbamoylase,which has low activity and poor thermostability,is the rate-limiting enzyme in the hydantoinase process.In our previous study,the catalytic efficiency of the D-carbamoylase NiHyu C has been enhanced by protein engineering,while the half-life of the best mutant M4 at 40°C is merely 1.3 h.In this thesis,the thermostability of NiHyu C-M4 was engineered,and the crystal structure and molecular mechanism were also analyzed.The main study contents and results are as follows:（1）The identification of key hot spots affecting the thermostability of M4.The ancestral sequence reconstruction of M4 was performed through Fire Prot^ASR website.Three ancestor sequence node sequences including 151,180 and 292 were obtained,and the evolutionary regions influencing the thermostability were identified.Based on the evolutionary regions,M4was compared with all ancestor sequences and 10 key hotspots were identified.Ten single mutants with improved thermostability were obtained by site-directed mutagenesis.Among them,R277L has a half-life of 8.0 h at 40°C,which is 6.3 times longer than that of M4,with significantly improved thermostability.（2）Combinatory mutagenesis and evolution analysis of thermostable mutants.The best mutant S202P/E208D/R277L（M4Th3）was obtained based on directional iterative combination mutation.The half-life of M4Th3 at 40°C increased to 36.5 h,which is 28.5 times that of M4.The k_cat/K_m of M4Th3 is 302 min^–1·m M^–1,which is higher than 288 min^–1·m M^–1 of M4.Through thermostability characterization and deconvolution analysis,it is believed that there is a significant synergistic/cumulative effect between S202P,E208D and R277L,while S204,E138 and H284 have antagonistic effects on R277L.（3）Elucidation of thermostability mechanism based on M4Th3 crystal structure and application study.The crystal structure of M4Th3（PDB ID:8I99）,a homotetrameric protein with space group P1,was obtained by sitting drop method.Molecular dynamics simulations of M4 and M4Th3 were carried out to elucidate the mechanism of their improved thermostability.S202P and E208D could expand the substrate channel,resulting in a higher enzymatic activity of S202P/E208D than that of M4,as well as significant increase in the number of hydrogen bonds,which improved the thermostability of the enzyme.R277L also leads to a slight increase in the number of hydrogen bonds,which is important for improving the stability of the subunit interface and contributes to a significant improvement in the thermostability of the enzyme.Using M4Th3,10 m M N-carbamyl-D-tryptophan was hydrolyzed to produce D-tryptophan.The conversion ratio reached 96.4%at 40°C for 12 h,significantly higher than 64.1%of M4.In this study,the thermostability of the D-carbamoylase mutant NiHyu C-M4 was engineered based on ancestral sequence reconstruction.The mechanism of its improved thermostability was elucidated based on crystal structure and molecular dynamics simulations,which could provide guidance for the engineering of D-carbamoylase and their application in the synthesis of D-amino acids.