Development of an efficient biocatalyst: engineered Burkholderia ambifaria lipase for aqueous esterification

Abstract

The aqueous esterification of isoamyl alcohol represents an environmentally friendly synthetic route. However, its industrial implementation is hindered by the scarcity of efficient catalysts, and conventional enzyme-directed evolution methods are often cumbersome and costly. To circumvent these limitations, this study employs the advanced protein design algorithm LigandMPNN for the rational design of lipase mutants. Using the aqueous esterification of isoamyl alcohol as a model reaction, we employed LigandMPNN to design mutations targeting the substrate-binding region. A key variant, Y181V, was created. Under optimized conditions, the mutant achieved a high yield of 217 mg/L isoamyl caproate. The Y181V variant exhibited 37% higher catalytic activity in aqueous phase than the wild-type. This mutation optimized the hydrophobic microenvironment of the active center, favoring the accommodation and transformation of hydrophobic substrates. This study successfully developed an improved lipase mutant for esterification, thereby validating the efficacy of LigandMPNN in protein rational design. Our work provides novel insights and a promising candidate for the creation of industrial-grade biocatalysts.

Keywords

Lipase / Esterification reaction / Rational design / Aqueous esterification

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Funding

the National Natural Science Foundation of China(No. 32571709)

the Research Program of State Key Laboratory of Food Science and Resources, Jiangnan University(No. SKLF-ZZB-202512)

RIGHTS & PERMISSIONS

Jiangnan University