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

Pei Yun Wang, Shau Wei Tsai*, Teh Liang Chen

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

34 Scopus citations


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 iso-octane 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/hg 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/hg 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/(E t) = l-56 mmol/hg and VS/VR = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/hg 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/hg 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/hg 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.

Original languageEnglish
Pages (from-to)460-469
Number of pages10
JournalBiotechnology and Bioengineering
Issue number3
StatePublished - 15 10 2008


  • (R,S)-2-hydroxycarboxylic acid esters
  • Covalent immobilization
  • Hydrolytic resolution
  • Substrate engineering


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