Study On The Building Of A Two-phase Partitioning Bioreactor For Enzyme-catalyzed Resolution Of Ketoprofen And Mandelic Acid

This thesis reviews resolution technologies for chiral drugs, and a novel two-phase partitioning bioreactor was built to separate the enantiomers of ketoprofen and mandelic acid with lipase catalysts.The polymeric microspheres of polysulfone containing n-hexanol, namely PSF/Hexanol, were prepared using the phase inversion method. The adsorption capability of the resulting PSF/Hexanol microspheres was tested using ketoprofen and mandelic acid, which were substrates for enzyme-catalyzed resolution. The results show that the diameter of the PSF/Hexanol microspheres was about2mm, and there were large number of macro and micropores inside the microspheres. The loading ratio of PSF/Hexanol for n-hexanol was80.70%. Also, the PSF/Hexanol microspheres showed good adsorption ability for both ketoprofen and mandelic acid. The highest adsorption ratio for ketoprofen and mandelic acid with different initial concentrations reached98.75%and92.23%in2.5h, respectively.A novel two-phase partitioning bioreactor (TPPB) was built using PSF/Hexanol microspheres, free porcine pancreas lipase (PPL), and phosphate buffer (KPB). Then the water soluble racemic ester of ketoprofen (EKS) we synthesized was asymmetrically hydrolyzed in TPPB to get (S)-Ketoprofen. Effects of pH, time, EKS concentration, PSF/Hexanol concentration on the conversion and enantiomeric excess of product were studied. Reactions in KPB water phase were also conducted to compare with the reactions in TPPB. The results show that in TPPB the optimal reaction pH was7.5, reaction time was24h, EKS concentration was2.5mg/ml and the PSF/Hexanol concentration was0.5g/ml. Also, compared to reaction in water phase, conversion in TPPB was higher.The two-phase partitioning bioreactor (TPPB) was built using PSF/Hexanol microspheres, immobilized enzyme Novozyme435(Candida Antarctica Lipase B, CAL-B), and phosphate buffer (KPB).Then the RS(±)-methyl mandelate was asymmetrically hydrolyzed in TPPB to get (R)-Mandelic acid. Effects of pH, time, temperature, methyl mandelate concentration, PSF/Hexanol concentration on the conversion and enantiomeric excess of product were studied. Reactions in KPB water phase were also conducted to compare with the reactions in TPPB. The results show that in TPPB the optimal reaction pH was6, reaction time was24h, temperature was40℃, methyl mandelate concentration was30mg/ml and the PSF/Hexanol concentration was0.3g/ml. Also, compared to reaction in water phase, the immobilized enzyme Novozyme435resulted in a better enantioselectivity in TPPB.A novel enzymatic-catalyzed resolution system, namely TPPB, for chiral drugs was made up in this thesis. On the one hand, TPPB provide enzymes a much milder catalytic environment which was essential for keeping enzyme activity. On the other hand, polysulfone microspheres were effective for separation and collection of optical products, as well as reusable for industrial applications.