Modification of the properties of the metagenomic lipase LipC12 by engineering of the hydrophobic cavity

Author(s)

Maldonado, Marcos Rodrigues ; Alnoch, Robson Carlos ; Shiratori, Gabriela Yumi Yamamoto ; de Oliveira, Cynthia Christina ; Goncalves, Marcos Brown ; Mitchell, David Alexander ; Krieger, Nadia

• Document type: Journal article
• Source: BIOCATALYSIS AND BIOTRANSFORMATION; v. N/A, p. 11-pg., 2023-05-02.

Abstract

Kinetic resolution of racemates with lipases is the preferred method for producing bioactive compounds. One strategy for obtaining highly enantioselective lipases that are stable in the organic media that are often used in these reactions is to improve existing lipases by protein engineering. In this work, we engineered the lipase LipC12, which has good stability in organic media, but only moderate enantioselectivity. Molecular docking with LipC12 identified V261 as a key position influencing, first, enantioselectivity in the transesterification of (RS)-1-phenylethanol and, second, activity in the hydrolysis of p-nitrophenyl octanoate. Variants were then obtained by site-directed mutagenesis, expressed in Escherichia coli, and their performance in these reactions was evaluated. Enzymes immobilized on Immobead 150 were used in the transesterification while free enzyme was used in the hydrolysis reaction. It was not possible to increase the hydrolytic activity and enantioselectivity simultaneously: some variants had increased enantioselectivity but lower hydrolytic activity, and others had increased hydrolytic activity but lower enantioselectivity. The best result for enantioselectivity was obtained for LipC12(V261Q), with an increase of the E-value (for (R)-1-phenylethanol) from 46 to 110, however, its hydrolytic activity decreased 4-fold in comparison to LipC12(wt). The highest hydrolytic activity was obtained for LipC12(V261F), with a value almost 6-fold higher than that of LipC12(wt). This variant also had an inverted enantiopreference (i.e. for (S)-1-phenylethanol), but with a very low E-value of only 4. (AU)

• FAPESP's process: 20/00081-4 - Development of strategies for immobilization and co-immobilization of enzymes involved in biomass degradation
• Grantee: Robson Carlos Alnoch
• Support Opportunities: Scholarships in Brazil - Post-Doctoral