Hydrocarbon synthesis from (waste)oils enabled by a cascade of lipase-catalysed hydrolysis and decarboxylase-catalysed decarboxylation has become an active area of research en route to alternative, biobased fuels. However, Poor substrate transport efficiency is a major issue causing low reaction rates. This study focused on a protein self-assembly strategy based on SpyTag/SpyCatcher to overcome diffusion limitations. For this, two fusion proteins, TLL-Linker-SpyCatcher based on the lipase from Thermomyces lanuginosus and CvFAP-Linker-SpyTag based on the fatty acid photodecarboxylase from Chlorella variabilis were designed. A covalent multi-enzyme complex (TLL-CvFAP) was formed spontaneously by self-assembly of each enzyme. The effects of temperature, pH and molar ratio of self-assembled components on assembly efficiency were investigated. The results showed that the multi-enzyme complex TLL-CvFAP reached about 60% after 12 h of assembly, and the enzyme activity of the multienzyme complex was increased by about 50% compared to that of the corresponding non-assembled enzymes. Under optimized conditions 10 mM soybean oil were converted into 25 mM of the corresponding hydrocarbons, suggesting a good potential of biofuel synthesis.

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A biocatalytic cascade transforming castor oil into (R,Z)-octadec-9-en-7-ol is presented by combining a lipase catalysed hydrolysis of castor oil into ricinoleic acid followed by a photoenzymatic decarboxylation into (R,Z)-octadec-9-en-7-ol. Conversion of up to 41.7% and overall product concentrations of up to 60 mM, this new bienzymatic and photocatalytic cascade exhibits significant potential for the valorisation of non-edible castor oil. The scope and limitations of the current system are described and discussed.@en

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

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A photobiocatalytic cascade transforming natural triglycerides into alkanes/alkenes is proposed. Starting from natural triglycerides, free fatty acids have been obtained using lipases. The free fatty acids were then, in a photoenzymatic step, decarboxylated into the C1-shortened alkanes using a recently described photodecarboxylase from Chlorella variabilis NC64A. This cascade produced alkanes from various natural (waste) oils in significant amounts (up to 24 g L⁻¹) and may provide a basis for valorisation of waste oils into a next generation of biodiesel.

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