Journal of Protein Chemistry

, Volume 14, Issue 4, pp 217–224

Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

  • Mats Holmquist
  • Ib Groth Clausen
  • Shamkant Patkar
  • Allan Svendsen
  • Karl Hult
Article

DOI: 10.1007/BF01886762

Cite this article as:
Holmquist, M., Clausen, I.G., Patkar, S. et al. J Protein Chem (1995) 14: 217. doi:10.1007/BF01886762

Abstract

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theVmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheKm,app for tributyrin was not affected by the Glu87Ala mutation, but theKm,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedVmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theKm,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theKm,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theVmax,app was only 6% of that obtained with wild-type enzyme.

Key words

Hydrolasesite-directed mutagenesiskineticscyclohexanebutanoic acid esters

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Mats Holmquist
    • 1
  • Ib Groth Clausen
    • 2
  • Shamkant Patkar
    • 2
  • Allan Svendsen
    • 2
  • Karl Hult
    • 1
  1. 1.Department of Biochemistry and BiotechnologyRoyal Institute of TechnologyStockholmSweden
  2. 2.Novo-Nordisk A/SBagsvaerdDenmark