The Control Of Enzymatic Enantioselectivity Of Lipase-catalyzed Hydrolysis Of Arylpropionate

The enzymatic reaction has played the important role in many fields. The control of the enzymatic reaction, especially the enzymatic enantioselectivity, has attracted much attention. Thus, in this thesis, the enzymatic enantioselectivity has been investigated and controlled through the change of enzymes, substrates and reaction media with the racemic temperature (Tr) and inversion temperature (Tinv). The enzyme immobilization has also been introduced into the enzymatic reaction to control the nature of the enzyme. It can offer an effective approach to the control of the enzymatic reaction for the applications in the organic synthesis and analytical chemistry.The racemic temperature (Tr) of the lipase-catalyzed hydrolysis of ketoprofen esters and the control of the enantioselectivity were investigated. Six lipases, including CRL, L-AYS, PPL, L-PS- L-AY30 and CCL, were used to catalyze the hydrolysis in aqueous. The Tr was different for different lipases. The lowest one was 28°C for PPL and the highest one was 51°C for L-AYS. The enantiopreference was S at T>Tr and R at T<Tr, representing an interesting reversal of the enzymatic enantioselectivity. The reaction media could also influence the Tr of the lipase-catalyzed hydrolysis of the ketoprofen vinyl ester. For example, in the L-AYS-catalyzed hydrolysis, the Tr could be decreased, even not existed, when the hydrophilic dioxane was added into the reaction medium; while the Tr could be increased when the hydrophobic diisopropyl ether (DIPE) and isooctane was added and thus the enantiopreference was R during 10～50°C without the reversal. For the ketoprofen alkyl ester hydrolysis catalyzed by CRL and L-AYS in aqueous, the Tr could be decreased with increasing chain length of the esters, from～20°C for methyl to the～-1°C for butyl. However, the enantiopreference could not be reversed from S to R when the T decreased below the Tr.Three solvents and four mixed reaction media was used to investigate the enzymatic enantioselectivity of the ketoprofen vinyl ester hydrolysis catalyzed by MJL. In aqueous, a Tr was obtained at 4.3°C, while, in DIPE and isooctane, the Tr could not be existent, and in the experiment temperature (10～50°C), the enantiopreference were all R-selectivity. However, in the four mixed reaction media, the non-linear Eyring plots were obtained with the Tinv. In the homogeneous mixed media of water and hydrophilic DMF (or acetone), the highest E value was shown at Tinv; while the lowest E value could be obtained at Tinv in the heterogeneous mixed media of water and hydrophobic DIPE (or isooctane). In the DMF/water homogeneous medium, Tinv could be decreased from>50°C to<10°C with increasing the ratio of DMF, and the Eyring plots during 10～50°C was changed from under the entropic control to the enthalpic control. In the DIPE/water heterogeneous medium, Tinv was about 25°C and could not be changed with the change of the ratios.For the control of the immobilization of penicillin G acylase (PGA), chitosan (Cs) was applied to modify the support and carrier. The immobilized PGA in the Cs-carrier has the highest volume activity (523 U/g) and specific activity (9.17 U/(g protein)), while the volume activity and specific activity of polyethyleneimine- and carboxylpropylchitosan-carrier were about 300 U/g and 5 U/(g protein). The specific activity of PGA immobilized on Cs-carrier was increased with increasing Cs molecular weight and decreasing Cs degree of deacetylation and the acetic acid concentration of the chitosan solution. Through the investigation of the horseradish peroxide adsorbed on different chitosan membranes, the results suggested that the amount of adsorbed enzyme was dependent on the network and structure of the membrane matrix. The apparent specific activity of adsorbed enzyme could be increased due to a low diffusional limitation of the small molecules of the enzymatic reaction in the chitosan membranes. Thus, the PGA immobilized on the novel composite carrier, Cs-cast D301 resin, could show a high specific activity (47.9 U/(mg protein)) and volume activity (1304 U/g). However, the application of different carriers for the immobilized PGA could hardly influence the enzymatic enantioselectivity of the hydrolysis for the ketoprofen vinyl ester and vinyl 2-phenylpropionate.