應用介質工程和基質工程改善木瓜脂肪分解酵素對(R,S)-naproxen氧酯之動力分割

ABSTRACT The papaya lipase stored in the crude papain from spay-dried latex of Carica papaya has been discovered as a versatile enentioselective biocatalyst to obtain chiral acids by using hydrolytic resolution of their racemic esters. The dogma of this dissertation is to improve the lipase-catalyzed hydrolysis of (R,S)-naproxen ester practised by adopting the enzyme screening, purification, medium engineering, and substrate engineering concepts. In terms of the enzyme screening and purification, we employed the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester via a crude Carica papaya lipase in water-saturated organic solvents as the model system, where effects of temperature and solvents on the lipase activity and enantioselectivtiy were studied. The optimal temperature of 60℃ for the maximum initial rate of (S)-ester with a high enantiomeric ratio (E = 122) in water-saturated isooctane is obtainable. Additionally, less hydrophobic solvents of cyclohexane and MTBE than isooctane are vital on decreasing the lipase activity and enentioselectivity. A kinetic analysis is further performed by using a Michaleis-Menten mechanism combined with the product inhibition and lipase deactivation, leading to good agreements of experimental and best-fitted conversions. Comparisons of enzyme performances for Carica papaya and Candida rugosa lipases indicate that the former is more enantioselective, active, and stable for (S)-naproxen ester. A crude lipase stored in Caraica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents. A comparison of the enzyme performances in terms of activity or enantioselective with those from Carica papaya lipase indicates that both enzymes display low tolerance to the hydrophilic solvent and are inhibited by (S)-naproxen or 2,2,2-trifluoroethanol. Improvements on the lipase activity and enantioselectivity are found when using both lipases in partially purified form as the biocatalysts. By utilizing the thermodynamic analysis, the enantiomeric discrimination is mainly driven by the difference of activation enthalpy for all reaction systems except for the hydrolysis of (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester employing Carica papaya lipases as the biocatalyst. As for medium engineering, the performance of pCPL for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated isooctane is altered when adding a variety of organo-soluble bases that act as either enzyme activators (i.e. TEA, MP, TOA. DPA, PY, and DMA), or enzyme inhibitors (i.e. PDP, DMAP, and PP). Triethylamine (TEA) was selected as the best enzyme activator as 2.24-fold increase of the initial rate for (S)-naproxen ester was achieved when adding 120 mM of the base. Furthermore, the activity enhancement for all lipases can also be observed, but with a negative effect on enantioselectivity, except for the partially purified lipase from Carica pentagona Heilborn as 60 mM TEA is added. By applying an expanded Michaelis-Menten mechanism for the acylation step, the kinetic analysis indicates that the proton transfer for the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is more affected than that for the formation of tetrahedral intermediates when adding an enzyme activator. However, no correlation for the proton transfer in the acylation step is found when adding the base acting as an enzyme deactivator. As for substrate engineering, with the hydrolytic resolution of (R,S)-naproxen ester via pCPL in water-saturated isooctane at 45℃ as the model system, the substrate containing a leaving group of different electron-withdraw capability has an extreme influence on the enzyme activity. About 630-fold enhancement of the initial rate for (S)-ester are obtainable when increasing the inductive parameter from 0.01 to 0.4 for the alcohol without containing a dimethylamino group. This implies that the acylation step must be the rate-limiting step. Meanwhile, we also assume that the bond-breaking of tetrahedral intermediate to acyl-enzyme intermediate may be the rate-limiting step for the compounds that alcohol group is hard to leave (e.g. 0.01 to 0.11). On the other hand, a novel means of using substrate-assisted catalysis indicates that the proton shuttle device acts as an effective tool for improving the lipase activity by forming an extra intramolecular hydrogen bond in the transition state. For the substrates with the same inductive parameter of 0.01 or 0.03, about 61-fold enhancement of lipase activity is obtainable. Moreover, about 80 ~ 90-fold increase of k2S/KmS implies the free energy difference of 11.5 ~ 11.9 kJ/mol between the transient states if one assumes the same ground states for both substrates. These values agree well with the stabilization by an additional hydrogen bond. Similar results for the activity enhancement are also found for using other lipases.