Thermal Stability Modification And Diastereoselectivity Improvement Of Pseudomonas Alcaligenes Lipase(PaL)

Lipase is a kind of biocatalyst with wide application.It has good stereoselectivity and activity for natural substrates,but it needs to be modified for activity,selectivity and stability when it is used for non-natural substrates.However,there is enzyme activity-stability trade-off phenomenon.How to maintain enzyme activity while improving its thermal stability has always been a challenge in enzymatic research.Previous studies have yet to provide a clear explanation for the non-additive mechanism of interaction between long-distance mutations,and predicting the effects of multiple mutations has always been a challenge for enzymatic research.Studies on diastereoselectivity applied to substrates containing multiple chiral centers are also rarely reported.The above-mentioned research is limited to enzymes which are synthezed by twenty kinds of natural amino acids,which limits the analysis of the mechanism of diastereoselectivity.In this thesis,Pseudomonas alcaligenes lipase(PaL)catalyzes the hydrolysis of the eight isomers of menthol propionate in the aqueous phase as a model to explore the mechanism of long-range mutations while maintaining enzyme activity and improving thermal stability.And to study the mechanism of improving diastereoselectivity by site-specific incorporation of unnatural amino acids with more functional groups in PaL.Details as follows:(1)Multi-strategy combination improves the thermal stability of PaL.Through the research of the wild type crystal structure of PaL obtained in previous work,the alignment with the lipase sequence derived from the thermostable strain,the calculation of the mutation energy of FireProt and the prediction of the soluble mutation of PROSS,three strategies were selected to screen out S78H,S205Y,Q289E and G379D for improved thermal stability.After the combined mutation of the four mutations,a 4M variant with improved thermal stability while maintaining enzyme activity was obtained.The protein melting temperature and the half-life at 50℃ of the 4M were increased by 14℃ and 15 times respectively compared with the wild type..(2)Employing Fitness Landscape to analyze non-additive effect on the 24 evolutionary paths generated by four mutations,and dynamic cross-correlation maps(Dynamics Cross-Correlation Maps,DCCMs)were used to analyze different mutation combinations.The interaction between the four long-range mutations and the effect of different mutation combinations on the entire protein were explored,the interaction relationship was found:the mutation S78H and the mutation S205Y cooperate with each other,and the mutation G379D interacts with S205Y and S78H through the active region 222-255.Mutations Q289E and S205Y mediated synergy through the active region 222-255.(3)By mutating the pyrrolysyl-tRNA synthetase to the N346A/C348A variant,the amber stop codon TAG was recognized incorporate 2-bromo-phenylalanine and 2-chloro-phenylalanine into PaL.pDule-CNF and pDule-aPhe were used to recognize TAG to incorporate 4-cyano-phenylalanine and 4-amino-phenylalanine into PaL,respectively.After the protein was purified,SDS-PAGE and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF-MS)were used to verify the site-specific incorporation of unnatural amino acids into PaL.Finally,when 4-bromo-phenylalanine was incorporated into position 253,variant A253Br with the best diastereoselectivity was obtained.When the conversion rate of L-menthol propionate>94%,its diastereomeric excess>95%,which is twice that of the wild type.(4)Employing covalently docking the substrate into PaL and then performing MD simulation,9 amino acid residues were located,which affected the three chiral centers.Four unnatural amino acids were incorporated into 9 positions in sequence,and the influence of the incorporation of unnatural amino acids at each position on the diastereoselectivity was analyzed.MD simulations found a linear relationship between the volume of amino acids incorporated at 253,the flexibility of the active center,and the diastereoselectivity.Therefore,how steric hindrance and flexibility affect diastereoselectivity were found.