Investigation On Molecular Engineering And Mechanism Of The Thermostability Of Alcohol Dehydrogenase KpADH

As natural catalysts,enzymes exibit great advantages including mild reaction conditions,high selectivity and atomic economy,in the industrial synthesis of chiral fine chemicals.However,most of the natural enzymes are suseptible to hash industrial conditions,such as high temperature,osmotic pressure and shearing force.Therefore,improving the thermostability of natural enzymes by molecular engineering has drawn increasing attentions.An NADPH-dependent short-chain alcohol dehydrogenase KpADH from Kluyveromyces polyspora has been successfully discovered with good performance in asymmetric reduction of?4-chlorophenyl?-?pyridine-2-group?-ketone?CPMK?into?S/R?-CPMA.However,the thermostability of KpADH is inadequate for application.In this study,a variety of molecular engineering strategies were employed to improve the thermostability of KpADH and elucidate the mechanism of its thermostability.The main research contents and results are as follows:Whole sequence random mutation library containing 8000 mutations of KpADH was constructed by error-prone PCR.Within the library,six single point mutants with improved thermostability were identified with T₅₀¹⁵ values increased by 1.5?1.9°C compared with wild type?WT?.Almost all the mutants displayed similar activity as WT except for K36E and S167A.Subsquently,a new strategy based on analysis of perturbation and free energy calculation was established?Rapid Enzyme Stabilization via Pertuabation and Energy Analysis,RESPEA?.Using this strategy,nine flexible regions of KpADH were determined by molecular dynamics simulation and then perturbation residues located in these regions were identified through residue interaction network?RIN?and centrality analysis.Finally,all the perturbation residues were virtually mutated to predict the changes in free energy and 24 point-mutations were predicted to be higher thermostable.Experimental verification showed that 10 of the 24 mutants,K36E/I,T132D,N159D,K210D/E,T248A,Q272F/L/M,showed enhanced stability.The most thermal stable mutants were mutant T248A and T132D,with T₅₀¹⁵ values of 45.8 and 44.7°C respectively,much higher than 42°C of WT.The seven residue sites identified above,K36,T132,N159,S167,K210,T248 and Q272,were saturationally mutateded to explore their evoloable patterns for thermostability.Furthermore,combinatorial mutation library was constructed by iterative strategy.After screening,KpADH_Th6h6 was obtained with the highest thermal stability.The T₅₀¹⁵ value of KpADH_Th6h6 was 53.1°C and the half-life at 45°C was 3000 min,which was 2700 times of that of WT.Kinetic parameter analysis showed that the k_cat/K_M of KpADH_Th6 toward CPMK was1.3 times of WT.In addition,the thermolstability fitness pathway landscape was constructed on the basis of the combinatorial mutation library.Through the landscape,epistatic interactions between mutation sites were analyzed.Unfortunately,the synergistic effect was not appeared during the combination.However,adverse pathways analysis disclosed antagonistic effects between T132D and N159D,providing guidance for molecular mechanism analysis.To further investigate the mechanism of KpADH thermostabiltiy,crystal structure of KpADH_Th6-NADP⁺complex was resolved.High-quality protein crystals in complex with NADP⁺were obtained through sessile drop method and the crystal data was collected by X-ray diffraction.Based on the crystal structure and MD simulation,the improvement of thermolstability of mutant Kp ADH_Th6 was highly correlated with the changed micro-perturbation of side chains of residues,which introduced new salt bridges or enhanced the local hydrophobic package.The antagonism interaction between mutant T132D and N159D was caused by the rotation of H134.Finally,the thermal stable mutant KpADH_Th6 was combined with the stereoselectivity reversed mutant KpADH_Mu-S5 to form mutant KpADH_Th6-S5.It can completely reduce 1.0 M CPMK to?S?-CPMA within 12 hours at 30°C.At 35°C,it can reduce500 mM CPMK within 4 hours,while the conversion rate of the control strain is<65%under the same conditions.This study provides guidance for molecular engineering the thermostability of ADH and its application in preparing of chiral compounds.