Rational Design And Modification Of Lipase/Esterase

Enzyme,a kind of biocatalysts,is widely distributed in nature.Due to its high catalytic efficiency,precise selectivity and environmental friendliness,enzyme is an ideal alternative for chemical catalysts and gradually becoming a hot spot for industrial and scientific researches.Owing to their origin,most of wild-type enzymes are sensitive to environmental changes and have narrow substrate spectra,making them difficult to carry out large-scale industrial applications.To this end,through enzyme engineering,we can get more stable and more widely used biocatalysts.So far,the main strategies to improve enzyme performance are immobilization,solvent engineering and protein engineering.Among them,protein engineering obtains physicochemical and molecular properties of proteins through protein chemistry,crystallography and kinetics.On these bases,the gene encoding the protein is purposely designed and modified.Subsequently,proteins with improved properties are obtained by genetic engineering and experimental validation.In protein engineering,there are three main categories,irrational design?random mutagenesis?,semi-rational design?site-directed saturated mutagenesis?and rational design?computer-aided point mutation?.Among them,irrational design and semi-rational design require high-throughput screening,which are more suitable for rapid-growing hosts and highly-expressed proteins.As structural and physicochemical data of proteins are soaring,rational design method processes protein information,designs proteins to meet the expected nature,and greatly reduces the experimental efforts.Thus,in this paper,via computer-aided rational design strategy,thermal stability of a lipase was greatly enhanced,and de novo design of esterase to catalyze specific substrate was further explored.The main contents and results were summarized below:1.The employment of single-point mutations to modify the thermostability of proteins is the most common means.There are a variety of computational algorithms that predict the effect of single-point mutations on thermostability.Before being used to rationally design thermal stability of proteins,computational algorithms should be evaluated to find the most suitable algorithm for predicting.Based on the data in Protherm database,various computer-aided predictors that predict stability change caused by single point mutation were evaluated.The Protherm database provides the effect of point mutations on the protein thermostability reported in the literature.In this study,point mutations were extracted from Protherm database as the test dataset.Accuracy and breadth of the prediction were treated as criteria to evaluate a variety of popular stability predictors.The results showed that three algorithms,I-Mutant 3.0,FoldX and Rosetta ddg_monomer,were excellent in the evaluation.Compared with the prediction results of single algorithm,the intersect results of the three algorithms?RIF?improved the accuracy to a certain extent.2.In order to evaluate the performance of RIF in enhancing thermostability of enzyme,Rhizomucor miehei lipase?RML?was selected for experimental validation.Through RIF,complemented with the removal of the sites that affect important functions of the enzyme,36 single-point mutants were finally constructed.Among them,24 mutants enhanced the thermal stability,and 13 mutants increased the apparent melting temperature(T_m^app)more than 1°C.The experimental results showed that combination of Rosetta ddg_monomer,I-Mutant 3.0 and FoldX could greatly reduce the experimental screening process and improve the prediction accuracy.In order to further improve the thermal stability of the enzyme,the additive effect of the single-point mutation was studied.The results showed that three single-point mutations,T18K?+4.5°C?,T22I?+2.4°C?and E230I?+5.7°C?,had a synergistic effect on improving the thermal stability.The T_m^apppp value of T18K/T22I/E230I was 9.4°C higher than that of wild-type RML.Compared to the wild-type RML,the optimum temperature of the triple mutant was increased by 10°C,and the half-life of the mutant at 70°C was increased by 5 times.Nevertheless,the optimal substrate?p-NP C8?and the optimum pH?8.0?did not change.3.Besides predicting the single-point mutations to increase the thermostability of the enzyme,rational design of disulfide bonds was also considered to further improve the thermal stability of RML.A number of potential amino acid pairs that can form disulfide bonds were obtained using four web-accessible tools?Disulfide by Design,MODIP,SSBOND,BridgeD?.The change of stability before and after mutation was calculated by FoldX.The residue pairs predicted to enhance stability were selected and visually inspected by YASARA.Finally,two potential disulfide bonds,S56C/N63C and V189C/D238C,were obtained for validation.The results showed that the T_m^apppp value of S56C/N63C and V189C/D238C mutation was further increased by 1.2°C and 4.2°C on the basis of T18K/T22I/E230I,while the mutant M7 containing the two disulfide bonds increased the T_m^app by 4.9°C compared to that of T18K/T22I/E230I and by 14.3°C compared to that of the wild-type RML.The studies of enzymatic properties showed that the optimum substrate for M7 was still p-NP C8,but the optimum pH of M7 was changed from 8.0 to 8.5.The optimum temperature of M7 was increased by 15°C,and the half-life of M7 at 70°C was increased by 12.5 times compared to that of the wild-type enzyme.4.Based on the above studies,the de novo design of an esterase to hydrolyze p-nitrophenyl acetate?p-NPA?was also explored.The conformation of catalytic residues and substrate in the active site?theozyme?was obtained via quantum chemical calculation,and a set of substrate rotamers was also constructed.Scaffold protein structures that can accommodate the theozyme and substrate rotamers were then searched.Amino acid residues near the active site were optimized to obtain an engineered enzyme that can hydrolyze target substrate.After filtration,the best designed enzymes were synthesized,expressed and purified for experimental validation.The experimental results indicated that the artificial enzyme based on 2EBD template was able to hydrolyze p-NPA.Through mutating residues in the active site and comparing the change of the catalytic activity before and after mutation,the relationship between the designed residues and the catalytic activity was confirmed.