PROBING A FUNCTIONAL-ROLE OF GLU87 AND TRP89 IN THE LID OF HUMICOLA-LANUGINOSA LIPASE THROUGH TRANSESTERIFICATION REACTIONS IN ORGANIC-SOLVENT

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 V-max,V-app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The K-m,K-app for tributyrin was not affected by the Glu87Ala mutation, but the K-m,K-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 V-max,V-app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the K-m,K-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 K-m,K-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 V-max,V-app was only 6% of that obtained with wild-type enzyme.