A fluorescence-based activity assay for immobilized lipases in non-native media

A new method for the analysis of lipase activity in the immobilized state is developed. The fluorescence assay aims to quantify the potential of lipases for the application in organic solvents. As lipases are universally immobilized on polymeric carriers for the use in bioorganic synthesis, the assay includes an immobilization step on the walls of polymeric cuvettes. The activity of the immobilized lipase is probed by 4-methylumbelliferyl ester hydrolysis. The activity retention as a function of solvent concentration is used as a measure for the solvent resistance of the enzyme variant. The method is applied to two different lipases, Candida antarctica lipase B (CalB) and Bacillus subtilis lipase A (BSLA) in the presence of the solvents acetonitrile and ethanol. By comparison of the assay results with a commercial biocatalyst consisting of CalB on polymeric carrier (Novozyme 435) it is demonstrated that the assay allows a good prediction of the activity of the respective lipase as immobilisate on polymeric carriers. The assay surpasses the respective analysis in solution in terms of accuracy and precision. [Display omitted] •A new lipase activity assay using in-situ adsorption of the enzymes on the walls of the analysis cuvettes is presented.•The assay determines the solvent resistance of immobilized lipases.•Fluorescence spectroscopy provides the sensitivity to analyze a single lipase monolayer.•The screening method allows the prediction of application areas of lipase variants as immobilized biocatalysts in organic synthesis.