Enhancement of Lipase CAL-A Selectivity by Protein Engineering for the Hydrolysis of Erucic Acid from Crambe Oil

• Published in: European Journal of Lipid Science and Technology
• Main Authors: Oroz-Guinea, Isabel, Zorn, Katja, Bornscheuer, Uwe
• Format: Article in Journal/Newspaper
• Language: English
• Published: 2019
• Subjects: biocatalysis; Candida antarctica lipase A; Erucic acid enrichment; lipid modification; protein engineering; Antarc*; Antarctica
• Online Access: https://zenodo.org/record/4264762; https://doi.org/10.1002/ejlt.201900115

The aim of this study is to pursue the identification and characterization of different CAL-A variants displaying higher specificity toward erucic acid than CAL-A wild type (wt). A careful analysis of the data generated from previously created site-directed saturation libraries reveals several variants that display a higher preference for the hydrolysis of p-nitrophenyl (pNP)-erucate over pNP-oleate than the wt. The best three candidates (CAL-A V238D, V238Y, and V286N) are applied in biocatalysis using both Crambe oil and ethyl ester derivatives. When acting on Crambe oil, these CAL-A variants are as efficient as CAL-A wt in terms of C22:1 enrichment and product recovery independently of the temperature (enrichment and recovery values between 70–76% and 67–79% at 37C, and between 71–73% and 61–75% at 50C). In contrast, hydrolysis of Crambe ethyl esters leads to substantially increased accumulations of C22:1 and recovery values (V238Y: 78% enrichment and 92% recovery; V286N: 83% enrichment and 91% recovery) when using CAL-A V238Y and CAL-A V286N compared to CAL-A wt (78% enrichment, 60% recovery) in the free fatty acid fraction. Practical Applications: This study describes the enhancement of lipase CAL-A selectivity for the isolation and recovery of erucic acid (C22:1) from plant oil or its ethyl ester derivatives. Hence, this approach could represent a more eco-friendly alternative for its application in processes where the erucic acid is used as building block, such as the production of surfactants or polymers.