Recombinant Expression,Structure Analysis And Substrate Specific Modification Of Thermophilic Lipase From Thermomicrobium Roseum

As a green biocatalyst,lipase is widely used in food,medicine,leather and chemical industry because of its excellent catalytic characteristics.However,most of the lipase enzymatic conversions in industrial processes occur in severe environments,for example,high temperature,nonaqueous phase,and extreme pH.Therefore,lipases which can meet the above conditions have aroused wide attention of researchers,especially the thermostability of lipases.A lipase from Thermomicrobium roseum DSM5159 was characterized based on the research in our lab.The expression system,crystal structure,heat resistant framework and catalytic efficiency were investigated.The main results and described as following:（1）In this study,a hypothetical lipase gene from Thermomicrobium roseum DSM5159 was recombinantly expressed and characterized in E.coli BL21 and B.subtilis W600,which were named TrLipE and TrLipB,respectively.The successful expression of TrLipE and TrLipB was further confirmed by peptide map fingerprinting（PMF）analysis.For both expression systems,the target enzyme revealed marked stability over a wide temperature and pH range.Additionally,the studied TrLipE and TrLipB was found to show remarkable tolerance in mixed systems constituted by water and organic solvents.Depending on the different expression systems,TrLipB has better enzymatic properties,in particular,thermostability and organic solvent tolerance.Based on the circular dichroism（CD）analysis,the secondary structural compositions were slightly different between TrLipE and TrLipB.The thermostability of TrLipE and TrLipB was further verified with nano-DSC analysis.The substrate specificity and enzymatic kinetics were analyzed as well.The studied TrLipE and TrLipB’s capability to catalyze p-nitrophenol esters with different carbon chain lengths was verified.Enzymatic transesterification of immobilized TrLipB was confirmed,with a molar conversion rate of 23.32%.This research therefore provides a candidate that could be applied for biocleanser production.（2）Based on excellent characteristics of TrLipB,the hanging-drop vapor diffusion method was used for the crystallization of TrLipB.After quality control and optimizing the crystallization conditions,TrLipB crystals were obtained and the crystallization conditions were:1 mol/L succinic acid,0.1 mol/L HEPES and 1%PEG2000 MME（w/v）pH 7.0.After the diffraction data were processed using program XDS,the resolution of the crystal was refined up to 2.0（?）,and the crystal belong to the group P43212.In addition,the crystal structure of TrLipB conforms to the typicalα/βhydrolase structure.According to the results of structure alignment analyses,the TrLipB showed the highest structural identity with the HSL family esterase（Est N7）from Bacillus cohnii strain N1.（3）To investigate the relationship between thermostability and proline content in the Loop region of TrLipB.The results showed that the thermostability of several substitutions decreased markedly,and with the increase of mutation sites,the thermostable framework was considerably further perturbed.However,the substitutions on the lid of the enzyme,including P49G and P48G,promoted the catalytic efficiency to approximately 130%.In MD simulations,the mutants showed high B-factors and RMSD values,whereas the secondary structures,radius of gyration,H-bonds,and solvent accessible surface areas of these mutants were markedly affected,which shown that Pro in Loop region plays an important role in thermostable framework of TrLipB.In addition,the optimal temperature and half-life under the optimal temperature of the chimeras obtained by the replacement of the lid was reduced than that of TrLipE.At the same time,the MD simulation results showed that the chimeras had lower thermostability,which indicated that the“lid”domain of TrLipE is also important for its thermostability.（4）The enzyme activity and substrate specificity of TrLipE chimeras were changed by the N-lid swapping between TrLipE and other lipases.The results shown that compared to wild TrLipE,18 chimeras shared a similar pH range and optimum pH.It could be found that different chimeras had different affinity（K_m）for different substrates.In terms of substrate specificity,the chimers TrL-2,TrL-3,TrL-17,TrL-18can specifically catalyze the substrate 4-nitrophenyl benzoate,of which TrL-17 has the highest k_cat/K_m value.In order to further improve the catalytic efficiency of TrL-17,the corresponding amino acid was mutated according to the results of molecular docking of TrL-17 with the substrate and the strategy of reducing the binding energy of enzyme and substrate complex.The results indicated that the substitution variants catalyzed the4-Nitrophenyl benzoate up to approximately 3.2-fold faster than the wild TrL-17.