Pei-Yun Wang

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)‐mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55°C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from VS/(Et) = 1.62 mmol/h g and VS/VR = 43.6 of (R,S)‐ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM‐87 from the producer) to 16.7 mmol/h g and 867 of (R,S)‐2‐methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)‐2‐hydroxycarboxylic acid esters, giving improvements of the enzyme performance from VS/(Et) = 1.56 mmol/h g and VS/VR = 41.9 of (R,S)‐ethyl 3‐chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)‐2‐methoxyethyl 3‐chloromandelate (16) for the immobilized enzyme, VS/(Et) = 5.46 mmol/h g and VS/VR = 8.27 of (R,S)‐ethyl 4‐chloromandelate (10) for free SNSM‐87 to 33.5 mmol/h g and 123 of (R,S)‐methyl 4‐chloromandelate (14) for the immobilized enzyme, as well as VS/(Et) = 3.0 mmol/h g and VS/VR = 7.94 of (R,S)‐ethyl 3‐phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)‐2‐methoxyethyl 3‐phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach. Biotechnol. Bioeng. 2008;101: 460–469.

(R,S)-2-chlorophenoxyl pyrazolides as novel substrates for improving lipase-catalyzed hydrolytic resolution.

The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 45°C were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azolides (1-4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)pyrazolide or (R,S)-α-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)pyrazolides (5-7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48) to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4-7 demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic contributions to the difference of Gibbs free energies. A thorough kinetic analysis further indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively.