To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a reversible process to become irreversible, to shift the equilibrium reaction in such a way that enantiopure compounds can be obtained from prochiral or racemic substrates, reduce or eliminate problems due to product inhibition or prevent the shortage of substrates by dilution or degradation in the bulk media, etc. In this review we want to illustrate the developments of recent studies involving in vitro multi-enzyme reactions for the synthesis of different classes of organic compounds.
Abstract:A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening five amino acid linker. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structure of the bifuctional enzyme has been analysed by CD spectroscopy, as well as that of the parental enzymes, in order to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Since S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centres in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channelling.
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%).
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