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

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.

Keywords:

• Lipases
• protein engineering
• chiral compounds
• kinetic resolution
• secondary alcohols

Author contributions

Marcos Rodrigues Maldonado, Robson Carlos Alnoch, and Gabriela Yumi Yamamoto Shiratori: experimental work and writing – original draft; Cynthia Christina de Oliveira, Marcos Brown Gonçalves: molecular docking studies; David Alexander Mitchell: conceptualization, writing – review and editing; Nadia Krieger: conceptualization, writing – review and editing, supervision.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Although funding was received from various research funding agencies (listed in the acknowledgements section), none of the funding agencies were involved in the design, execution or reporting of the work.

Additional information

Funding

Research scholarships were granted to Marcos Maldonado by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), a Brazilian government agency for the development of personnel in higher education, and to David Mitchell and Nadia Krieger by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), a Brazilian government agency for the advancement of science and technology. Robson Alnoch received a postdoctoral fellowship [Grant No: 2020/00081-4] from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo). The Postgraduate Program in Science - Biochemistry of the Federal University of Paraná is financed, in part, by CAPES (Finance Code 001).