Biocatalysis Asymmetric Synthesis Of (R)-3-Quinuclidinol

(R)-3-quinuclidinol is an important chiral intermediate in several new types of medicines,including some kinds of new anticholinergic drugs for treating Chronic Obstructive Pulmonary Disease,such as Aclidinium bromide and Revatropate,Solifenacin to treat Overactive Bladder and Talsaclidine to treat Alzheimer's disease.Whether chemical methods,such as chemical resolution method,chemical asymmetric synthesis method,chiral chromatography method,or biological methods such as enzymatic resolution method,biosynthesis and biological resolution method,the final product(R)-3-quinuclidinol enantiomer value is not high.Therefore,in this experiment,we take advantage of Gene Recombination Technology,Protein engineering,and Permeabilization Technology,to establish a green and high efficient biocatalysis method to synthetise(R)-3-quinuclidinol.Specific contents are as follows:1.The construction of engineering bacteria.According to the nucleotide sequences of Quinuclidinone Reductases(QNR)and Glucose Dehydrogenase(GDH)in genbank,obtained gene fragments by synthetic method,connected with p ET-28? by double digestion,and constructed the plasmids p ET28?-QNR and pET28?-GDH,which are transformed into E.coli DH5? and E.coli BL21,then used for the plasmids extraction and enzyme expression.2.The expression of enzyme and optimization of reaction condition.In order to obtain the target protein with high expression and soluble,we would optimize the expression condition,such as the culture temperature,the concentration of Isopropy-?-D-Thiogalactoside(IPTG)and induction time.Finally we found the optimum culture condition by SDS-Polyacrylamide Gel Electrophoresis analysis(SDS-PAGE)according to the dgree of expression and catalysis: the temperature is 25 ?,the final concentration of IPTG is 0.8 mM and the best induction time is 72 h.3.In situ purification and immobilization enzyme.By the method of Immobilized Metal Ion Affinity Chromatography(IMAC)to achieve the situ purification and enzyme immobilization.After purification,the concentration of QNR is 0.557 mg/mL,enzyme activity is 4.2 U/mL.And the concentration of GDH is 0.600 mg/mL,enzymatic activity is 10.8 U/mL.4.Biocatalytic asymmetric synthesis of(R)-3-quinuclidinol.The synthesis of(R)-3-quinuclidinol was based on the whole-cell,permeabilized-cell and immobilized enzyme technology.Using the whole-cell as catalysis to optimize the synthesis condition,such as temperature,p H and the ratio of catalysis to substrates.The optimum temperature is 25-30 ?,pH 7-8,the proportion of whole-cell to the maximum substrate is 1:13,and the conversion rate is 100 % by Gas Chromatography.Fermentation volume is 50 g/L.Using permeabilized cells as the catalyst,toluene as the permeability agent,the amount of toluene is 0.4 % of total volume,and the other conditions are the same as the whole-cell.We found that the reaction time can be shorten 0.5-1 h.Immobilized enzyme as catalyst,while the ratio of substrate to immobilized enzyme is 9:1,the conversion time is 3 h,conversion rate is 100 %.When the ratio of substrate to immobilized enzyme is 11:1,the conversion time is 4 h,conversion rate is 100 %.When the ratio of substrate to immobilized enzyme is 13: 1,the conversion time is 5 h,conversion rate is 100 %.When the ratio of substrate to immobilized enzyme is exceeded 14: 1,the substrate can not be completely converted.With the repeatedly cycles using of immobilized enzyme while using the immobilized enzyme as catalyst tofinish the cycle reaction,the reaction time increased,and the immobilized enzymatic activity reduced.The amount of the substrate is 5.5 times to the immobilized enzyme,it can be recycled five times,the conversion rate is 100%.The amount of the substrate is 3.5 times to the immobilized enzyme,it can be recycled seven times,the conversion rate is 100%.And the maximum number of cycles is eight times.5.Isolation and purification of(R)-3-quinuclidinol.Precipitation protein with TCA,filtration to obtain a clear filtrate after,and adjust the pH with NaOH until the pH?12,then obtain the white solid by concentration.Dissoved the white solid with hot toluene and filtration immediately to remove insoluble remaineder,then cooling-crystallization to obtain the white needles crystal,the final yield is 76 %.6.Structure confirmation of(R)-3-quinuclidinol.mp: 217-224 ?,Via 1H-NMR and MR structural analysis method,the product is 3-quinuclidinol.The product configuration is confirmed to be(R)-3-quinuclidinol by GC analysis technology,and ee is100%.From the experiment we know that whether using whole-cell,permeabilized-cell or immobilized enzyme as biocatalyst,biocatalysis asymmetric reduced 3-quinuclidinone could obtained(R)-3-quinuclidinol in a high yield,high conversion rate and high optical activity.At the same time we get rid of using a large amount of organic solvent,which provided theoretical and technical support for green and industrialed synthesis of(R)-3-quinuclidinol providing.