Development of Biocatalysts Using Organized Nano-reactors

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Kinetic resolution of 1,1-diphenyl-2-propanol was performed at high temperatures using a lipase immobilized on porous ceramics, Toyonite 200M. Reactions proceeded even at 120℃, giving enantiomerically pure ester product. In contrast, the reaction at 100℃ using the same lipase immobilized on Celite resulted in no reaction. The high-temperature biocatalysis in supercritical CO_2 medium at 80℃ gave 42% conversion with an E value of 211. The lipase-catalyzed acylation of a secondary alcohol having tetraphenylporphyrin as a substituent proceeded at 60℃ most efficiently, giving enantiomerically pure ester and alcohol.Lipase mutant was immobilized on the porous ceramics to study how far the enantioselectivity can be controlled on the basis of a reaction mechanism. Substitution of some amino acid residue in the proximity of the active site with another one resulted in a change in enantioselectivity, as expected. Thermodynamic parameters suggested that the change in enantioselectivity was driven by the change in activation enthalpy.A new method for the prediction of absolute configurations of secondary alcohols using immobilized lipase was invented. Using the E values for two kinds of 1-substituted ethanols having different substituents, the E value for another secondary alcohol having the two substituents on both sides was predicted and determined. The experimental results for 19 out of 20 examples were consistent with the prediction.A carbonyl reductase obtained with recombinant E.coli was immobilized on the porous ceramics, which unfortunately showed no remarkable effect. Rather, whole-cell reductions using E.coli coexpressing two genes encoding the carbonyl reductase and a glucose dehydrogenase gave optically active alcohols much more easily and efficiently. Highly enantioselective asymmetric reductions were achieved, and 8 out of 16 ketones were reduced to optically active alcohols with >98% ee.