Enzymes are commonly immobilized in mesoporous silica particles in order to simplify the recovery of the proteins from the reaction mixture so they can be used again. Here we investigate the immobilization of two different types of enzymes (lipase and glycerol kinase) which together form a catalytic cascade, in the sense that the product of the first enzyme (glycerol) is the substrate of the second one. Using UV-vis spectroscopy tools developed previously we investigate if co-localization of the two enzymes in pores of similar dimensions as the proteins themselves facilitates the formation of the final product, as can be anticipated because the glycerol-intermediate will be produced by the first enzyme in close proximity to the second one due to the confining environment. Covalent dye-labelling of the two proteins are performed by established protocols, known not to interfere with the enzymatic activity. The labeled proteins are used to measure immobilization rates and average concentration of the immobilized proteins by emission spectroscopy, the degree of co-localization by fluorescence detected flow cytometry analysis of individual particles, the average distance between the two types of proteins in the pores by fluorescence resonance energy transfer, and the global distribution of the proteins in the particles by super-resolution fluorescence microscopy and second ion mass-spectroscopy imaging. Together these techniques can be used to answer critical question regarding the two-enzyme system, such as where are the proteins localized in the pores and how close are they to each other and the pore mouth. The last issue is central since the triglyceride substrate of the first enzyme (lipase) is not soluble in water and will remain in the solution surrounding the particles, whereas both enzymes and the glycerol intermediate are and hence are hypothesized to remain in the silica particle pores which are filled with water. Such a strategy with the enzyme meeting the substrate at the mouth of the particle pores has been successful in single-enzyme systems based on lipases or feruloyl esterases. Increasing the enzyme-catalytic efficiency of a two-step reaction from a triglyceride to a modified glycerol is attractive from a sustainability point of view, because the methyl ester produced in the first step is commonly used as a biofuel, and derivatives of the byproduct glycerol (in our model system glycerol phosphate) have promising properties for instance as green solvents.
Biträdande professor at Chemistry and Chemical Engineering, Physical Chemistry
Funding years 2016–2019
Area of Advance
Chalmers Driving Force