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

Abstract

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 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 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 V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 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, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 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 V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 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.

Rational design of esterase BioH with enhanced enantioselectivity towards methyl (S)-o-chloromandelate

“…However, the present hydrolases reported showed low activity and/or enantioselectivity towards (R,S)-CMM, which might be closely related with the sterically hindered 2-chlorophenyl moiety (Wang et al 2006b;Wang et al 2008). For example, the Candida antarctica lipase A catalyzed transesterification of (R,S)-CMM using vinyl propionate as an acyl donor with an enantiomeric ratio (E) of 34.7 (Uhm et al 2007).…”

“…In the present work, we aimed to develop an efficient strategy to improve the enantioselectivity of the existing enzymes towards (R,S)-CMM. It was reported that the 2-substituent of the acyl part strongly affected the rate-limiting formation or breakdown of the tetrahedral intermediate, and the stabilization of the transition state for the formation of the tetrahedral intermediate of the fast-reacting esters might increase the enzyme activity and enantioselectivity (Wang et al 2008). Therefore, herein, we speculated that the enzymatic enantioselectivity might be efficiently enhanced through manipulating the transition state by rational design.…”

“…Though a covalent immobilized enzyme was prepared to further improve the activity and enantioselectivity of the K. oxytoca esterase towards 2-hydroxycarboxylic acid esters, this strategy seemed not suitable for (R,S)-CMM as the immobilized enzyme Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5995-x) contains supplementary material, which is available to authorized users. exhibited remarkably reduced activities (Wang et al 2008). In the present work, we aimed to develop an efficient strategy to improve the enantioselectivity of the existing enzymes towards (R,S)-CMM.…”

Immobilization ofBacillus licheniformisL-Arabinose Isomerase for Semi-ContinuousL-Ribulose Production

“…Enzyme loading has been found to differ from enzyme to enzyme. For example, for lipase or penicillin acylase, enzyme loading was 100 mg/g 15) and 40 mg/g support 12) respectively. Compared with these two enzymes, the enzyme loading of BLAI was low, but was higher than that of penicillin V acylase (1 mg/g of support), 16) and was similar to that of glucose isomerase.…”

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

“…The good enthalpyentropy compensation relationships of DS R 5 2113. 5 . Thus, the entropic compensation effect is nearly negligible at 458C for obtaining an excellent CALB enantioselectivity.…”