More efficient enzymatic cascade reactions by spatially confining enzymes via the SpyTag/SpyCatcher technology

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 Cv FAP-Linker-SpyTag based on the fatty acid photodecarboxylase from Chlorella variabilis were designed. A covalent multi-enzyme complex (TLL- Cv FAP) 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- Cv FAP 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.