Alteration of Chain Length Selectivity of Candida antarctica Lipase A by Semi‐Rational Design for the Enrichment of Erucic and Gondoic Fatty Acids

Abstract

Biotechnological strategies using renewable materials as starting substrates are a promising alternative to traditional oleochemical processes for the isolation of different fatty acids. Among them, long chain mono‐unsaturated fatty acids are especially interesting in industrial lipid modification, since they are precursors of several economically relevant products, including detergents, plastics and lubricants. Therefore, the aim of this study was to develop an enzymatic method in order to increase the percentage of long chain mono‐unsaturated fatty acids from Camelina and Crambe oil ethyl ester derivatives, by using selective lipases. Specifically, the focus was on the enrichment of gondoic (C20:1 cisΔ11) and erucic acid (C22:1 cisΔ13) from Camelina and Crambe oil derivatives, respectively. The pursuit of this goal entailed several steps, including: (i) the choice of a suitable lipase scaffold to serve as a protein engineering template (Candida antarctica lipase A); (ii) the identification of potential amino acid targets to disrupt the binding tunnel at the adequate location; (iii) the design, creation and high‐throughput screening of lipase mutant libraries; (iv) the study of the selectivity towards different chain length p‐nitrophenyl fatty acid esters of the best hits found, as well as the analysis of the contribution of each amino acid change and the outcome of combining several of the aforementioned residue alterations and, finally, (v) the selection and application of the most promising candidates for the fatty acid enrichment biocatalysis. As a result, enrichment of C22:1 from Crambe ethyl esters was achieved either, in the free fatty acid fraction (wt, 78%) or in the esterified fraction (variants V1, 77%; V9, 78% and V19, 74%). Concerning the enrichment of C20:1 when Camelina oil ethyl esters were used as substrate, the best variant was the single mutant V290W, which doubled its content in the esterified fraction from approximately 15% to 34%. A moderately lower increase was achieved by V9 and its two derived triple mutant variants V19 and V20 (27%).

Enrichment of Erucic and Gondoic Fatty Acids from Crambe and Camelina Oils Catalyzed by Geotrichum candidum Lipases I and II

“…The following assays were performed as previously described: (1) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), (2) protein concentration quantification, (3) determination of the melting temperature, and (4) selection of the best P. pastoris clones (Oroz‐Guinea et al, ; Zorn et al, , b).…”

“…Subsequently, 5–10 μg were used to transform P. pastoris GS115‐competent cells by electroporation as described by Invitrogen. From each transformation, 20 colonies were picked, and the best P. pastoris clones were selected by screening the Zeocin resistance and the hydrolytic activity of the secreted lipases on indicator plates as previously described (Kouker and Jaeger, ; Zorn et al, , b).…”

“…Consequently, several studies analyzing the selectivity of lipases toward different FA and their potential use for the enrichment of gondoic and erucic acids were described. Recently, the creation of Candida antarctica lipase A (CAL‐A) variants was reported by our group as being able to enrich long‐chain mono‐unsaturated FA (MUFA) by either (1) enhanced selectivity toward the hydrolysis of FA shorter than 20 carbon atoms (Zorn et al, , b) or (2) increased tendency for their hydrolysis (Oroz‐Guinea et al, ). Thus, these approaches led to the enrichment of the longer FA in the esterified FA (EFA) fraction or in the FFA fraction, respectively.…”

“…For this purpose, the suitability of both lipase isoforms to access enriched FA fractions was investigated in hydrolysis and ethanolysis reactions. In addition, as it was previously observed that the lipase selectivity can be affected by the type of ester substrate used (Oroz‐Guinea et al, ; Zorn et al, , b), during the hydrolysis reactions, two formulations were used, the corresponding TAG and the FAEE derivatives.…”

Recent advances in high-throughput mass spectrometry that accelerates enzyme engineering for biofuel research

“…For example, surrogate ester substrates are utilized for rapid profiling of lipase activities by monitoring the release of p-nitrophenol at 405 nm upon ester hydrolysis (Fig. 3) [58][59][60]. The specificity of a select lipase can be characterized using a panel of surrogate esters with different fatty acyl chains.…”