The Research Of Pseudomonas Alcaligenes Lipase Modification And The Resolutive Preparation Of L-menthol

l-menthol is a kind of important chiral chemical. It has been used extensively in industrial and pharmaceutical field due to its unique flavor, cooling and refreshing effects. The yield and quality of natural menthol are affected seriously by weather and region. Moreover, due to the rapidly increasing demand for l-menthol among the global market in recent years, the l-menthol from natural sources has no longer satisfied the demand. How to apply biocatalysis method to produce l-menthol has became the research focus gradually.A strain named Pseudomonas alcalignens CGMCC4405producing lipase with high selectivity and hydrolysis activity toward the mixture of8diastereomers of racemic menthol to produce l-menthol was isolated from soil. The Pseudomonas alcaligenes lipase (PaL) gene was cloned and expressed in E.coli by the construction of genomic library. After PaL purification, the result of MALDI-TOF mass indicated that the precise molecular weight of PaL was58094.3Da and the peptide mass fingerprinting verified the amino acid sequence deduced from DNA sequence. The result of far-UV CD showed that PaL had26.8%a-helix,34.2%p-sheet,14.2%turn and24.7%random coli. The PaL had high enantioselectivity (E>200) in the process of resolution. However, the diastereoselectivity was not very ideal. The enzyme activity in shake flask was4625U/L. The optimal temperature and pH of PaL was35℃and9.0respectively. The PaL had good pH stability. Conversely, the thermal stability was not ideal.Contrary to the property of excellent enantioselectivity, we investigated the enantiomers recognition mechanism of PaL. Based on the analysis of the structure of PaL-substrate complex, the orientation of isopropyl connected with C2stereo center of d-menthyl propionate towards the imidazole ring of catalytic residue His271was identified. The steric exclusion effect of isopropyl forced the imidazole ring to deflect30°. The deflection caused the distance between Hs(His271) and O(alcohol) increased from2.2A to3.7A, which was unable to form the essential hydrogen bond. The lack of the essential hydrogen bond caused the PaL-d-menthyl propionate became not stable enough. Compared to l-menthyl propionate, d-menthyl propionate couldn’t be hydrolyzed normally. This may be the structure basis of excellent enantioselectivity of PaL.To resolve the problem of low diastereopreference, the interaction modes between PaL and substrate were investigated by molecular modeling and covalent docking. Under the guidance of the result of covalent docking, the double sites mutation V180L/A272F was constructed. The diastereomeric ratio of l-menthol hydrolyzed by variant PaL towards l-neomenthol increased from6.6:1of WT PaL to30.7:1. For the diastereomeric ratio of d-isoneomenthol, it increased from12.0:1to25.5:1. The result of molecular dynamic simulation indicated that stronger steric exclusion effect was introduced to certain location of PaL by site-directed mutagenesis. The increased steric exclusion effect restricted the orientation of group connected with stereo centers. The increased the structure rigidity of the region around the mutation sites consolidated the restriction effect of steric exclusion. Finally recognition specificity of substrates with different configurations and the diastereoselectivity were enhanced.The combined method of site-directed mutagenesis and chemical modification was employed to further improve diastereoselectivity of PaL. The strategy involved the introduction of a single cysteine residue into the hot spot via site-directed mutagenesis to constructe the variant A272C. This was then covalently linked with DTNB to give chemically modified mutant lipase. The result of MALDI-TOF mass verified the effectiveness of DTNB modification. The far and near-UV CD demonstrated the secondary and tertiary structure of PaL did not changed obviously after DTNB modification. The analysis of kinetic properties and molecular dynamic simulation showed that when bound with l-menthyl propionate, WT and A272C-DTNB modified PaL exhibited similar Km values. In contrast, the binding of non-target substrates produced increased Km values due to decreased flexibility and increased steric exclusion. The significant correlation of PaL flexibility with the Km value of the non-target substrate can be explained that the increased steric exclusion introduced by DTNB-modification of Cyc272and encountered by non-target isomers prevents their fitting into the PaL’s active site by restricting the orientation of the isopropyl group of the substrate.In order to achieve the effective preparation of PaL, we investigated the high-density fermentation of E. coli. The yield coefficient of glucose towards dry cell weight (Yx/s) is0.7. In the fed-batch fermentation stage, exponential feeding was used to control the specific growth rate at0.21h-1. The IPTG (20μmol/g dry cell weight) was added to fermentation broth in the15th hour at20℃. In the end of fermentation, the maximum dry cell weight is70g/L. The maximum enzyme activity of PaL is200000U/L. The result of SDS-PAGE gel electrophoresis showed that the PaL expressed effectively as soluble protein. Almost no inclusion body formed.On the basis of high-density fermentation, the recombinant E.coli whole cells were used to catalyze the hydrolysis of recemic menthyl propionate to produce/-menthol. Some key parameters of the biocatalytic process, including substrate and product inhibition, reaction temperature, catalyst loading, substrate loading and amount of cosolvent were optimized. The optimal biocatalytic process parameters were:reaction temperature30℃, catalyst loading2U/mL, substrate loading1000mM (21.2%, w/v) and cosolvent concentration5%. Under this optimal condition, the productivity of1.54gL-1h-1was achieved. The dep of target product/-menthol obtained by distillation was87.4%and the purity is90.45%. The total yield of l-menthol achieved88.24%。 These results indicate that the biocatalytic method has great industrial application prospect in the manufacturing of l-menthol.