Formation of enzyme-surfactant nanoconjugates for non-aqueous production of alkene biofuels from triglycerides Jake Nicholson 1 , Leticia M. Zanphorlin 2 , Alex P.S. Brogan 1 1 King's College,UK, 2 Laboratório Nacional de Biorrenováveis, Brazil Biofuels are important alternatives to fossil fuels as they are derived from renewable sources, such as biomass, instead of oil. Biodiesel is typically produced by transesterification of bioderived triglycerides with short-chain alcohols. Unfortunately, due to their high oxygen content, the widespread uptake of biodiesel has been slow due to incompatibility of existing engines requiring either the engines to be modified or the biofuel to be blended with petrodiesel 1 . Recently, biocatalytic pathways have been developed which have allowed for the production of alkanes and terminal alkenes from triglycerides. These hydrocarbon products are similar in composition and properties to petroleum derived fuels and hence can be used as drop-in fuels in existing infrastructure. Typically, biocatalytic pathways use a lipase to cleave the triglyceride to from a free fatty acid which is then decarboxylated to form a hydrocarbon 2 . However, these systems are limited by the low solubility and low critical micelle concentration of the substrates in aqueous media which is typically required for biocatalytic reactions. Previous work by Brogan et al. has focused formation of enzyme-surfactant nanoconjugates which have been shown to form solvent-free biofluids which retain enzymatic activity in the solvent-free state 3 and in ionic liquids 4 . The tuneable polarity of ionic liquids allows for a significant increase in the solubility of the substratesand the nonvolatility of ionic liquids may facilitate product separation by distillation. Here, we will present recent work on the formation of nanoconjugates of a lipase and a P450 fatty acid decarboxylase for non-aqueous biocatalytic production of drop-in biofuels. The resulting nanoconjugates were shown to have increased thermal stability, comparable aqueous activity to the unmodified enzyme, and were soluble in both the substrate and a range of ionic liquids. Preliminary data has shown that the Soret band of the P450 responds to illumination with UV light when the P450 nanoconjugate is dissolved in ionic liquid indicating the illumination is causing a photochemical reaction to occur. When this system was tested with the substrate present there was significant consumption of the substrate compared to a control which was kept in the dark. Future work will focus on optimisation of the photobiocatalytic formation of terminal alkenes from triglycerides in ionic liquids. References 1. Mishra VK, Goswami R. A review of production, properties and advantages of biodiesel. https://doi. org/101080/1759726920171336350 . 2017;9(2):273-289. doi:10.1080/17597269.2017.1336350 Xu W, Mou K, Zhou H, Xu J, Wu Q. Transformation of triolein to biogasoline by photo-chemo-biocatalysis. Green Chem . 2022. doi:10.1039/D2GC01992B 2. Brogan APS, Sharma KP, Perriman AW, Mann S. Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °c. Nat Commun . 2014;5(1):1-8. doi:10.1038/ncomms6058 3. Brogan APS, Bui-Le L, Hallett JP. Non-aqueous homogenous biocatalytic conversion o polysaccharides in ionic liquids using chemically modified glucosidase. Nat Chem . 2018;10(8):859-865. doi:10.1038/s41557-018-0088-6
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