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

Abstract

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 37 C, and between 71-73% and 61-75% at 50 C). 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.

Efficient and selective catalytic hydroxylation of unsaturated plant oils: a novel method for producing anti-pathogens

“…It can be rationalized by the solubility and fact that there is no extra oxidizing source in the reaction mixture to facilitate the formation of the Fe (III)(citrate)-Na moiety. In this case, may not be realized (S 2 O 8 ) 2− to initialize the [ 9 , 11 ]-hydrogen shift mechanism, while the Fe(III)/Fe(II) catalytic cycle for the [ 9 , 12 ]-hydrogen shifts [ 38 ].…”

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).…”

“…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.…”

Conversion of fatty acid methyl esters into dibasic esters by metathesis and their lubricant properties

“…It should be noted that biocatalytic pathways to selectively hydrolyze and transesterify erucic a. 30 or convert erucic a. in to a,u-C22:1 have already been reported, 31 which provides one more incentive to fractionate FAME and produce dibasic esters for HF. Another option to achieve VG32 or VG46 viscosities might be esterication with other alcohols, heavier than 2EH.…”