title: Probing a functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase through transesterification reactions in organic solvent.
abstract: To reveal the functional role of Glu87 and Trp89 in the lid of Humicola 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 the Vmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The Km,app for tributyrin was not affected by the Glu87Ala mutation, but the Km,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 decreased Vmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the Km,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 the Km,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 the Vmax,app was only 6% of that obtained with wild-type enzyme.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Holmquist
authors.forename: I G
authors.initials: IG
authors.lastname: Clausen
authors.forename: S
authors.initials: S
authors.lastname: Patkar
authors.forename: A
authors.initials: A
authors.lastname: Svendsen
authors.forename: K
authors.initials: K
authors.lastname: Hult
mesh_terms: D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism; D003510:Cyclohexanes; D004951:Esterification; D005973:Glutamine; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012997:Solvents; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism; D014364:Tryptophan; D014753:Vinyl Compounds / Q000378:metabolism
chemical_list: D002087:Butyrates; D003510:Cyclohexanes; D012997:Solvents; D014280:Triglycerides; D014753:Vinyl Compounds; D005973:Glutamine; C095498:vinyl butyrate; D014364:Tryptophan; D008049:Lipase; C005830:tributyrin