This review first provides a brief introduction into the most important tools and strategies for protein engineering (i.e. directed evolution and rational protein design combined with high-throughput screening methods) followed by examples from literature, in which enzymes have been optimized for biocatalytic applications. This covers engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity and improved performance in esterification reactions. Furthermore, recent achievements reported for phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, fatty acid hydratases and the use of enzymes in cascade reactions are treated.

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%).

No abstract

Acquisition of drug-reinforced behavior is accompanied by a systematic increase of release of the neurotransmitter acetylcholine (ACh) rather than dopamine, the expected prime reward neurotransmitter candidate, in the nucleus accumbens core (AcbC), with activation of both muscarinic and nicotinic ACh receptors in the AcbC by ACh volume transmission being necessary for the drug conditioning. The present findings suggest that the AcbC ACh system is preferentially activated by drug reinforcers, because (1) acquisition of foodreinforced behavior was not paralleled by activation of ACh release in the AcbC whereas acquisition of morphine-reinforced behavior, like that of cocaine or remifentanil (tested previously), was, and because (2) local intra-AcbC administration of muscarinic or nicotinic ACh receptor antagonists (atropine or mecamylamine, respectively) did not block the acquisition of food-reinforced behavior whereas acquisition of drug-reinforced behavior had been blocked. Interestingly, the speed with which a drug of abuse distributed into the AcbC and was eliminated from the AcbC determined the size of the AcbC ACh signal, with the temporally more sharply delineated drug stimulus producing a more pronounced AcbC ACh signal. The present findings suggest that muscarinic and nicotinic ACh receptors in the AcbC are preferentially involved during reward conditioning for drugs of abuse vs sweetened condensed milk as a food reinforcer.

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.