Certain lipases such as Candida antarctica lipase A (CAL-A) are known to possess acyltransferase activity, which allows ester synthesis even in the presence of water. Discovery of novel lipases and identification of lipase variants with improved acyltransferase activity is laborious using standard methods such as gas chromatography. To overcome this limitation, an enzyme cascade was established based on quantification of the alcohol consumed in fatty acid ester synthesis from plant oils. For this, an alcohol oxidase converts the remaining alcohol to the corresponding aldehyde with concomitant formation of hydrogen peroxide. This is then quantified by the action of horseradish peroxidase leading to the oxidation of the co-substrate ABTS, which is determined spectrophotometrically at 695 nm. The conversions thus determined correspond well to data measured by off-line gas chromatography analysis and the assay could be adapted to the microtiterplate format.Practical applications: Fatty acid esters are valuable compounds in various industrial applications, i.e., fatty acid methyl esters are used as biofuels and ethyl esters can serve as fragrance or flavoring compounds in the cosmetic industry. The enzymatic synthesis of these compounds is possible, but often enzymes need to be adapted to meet process requirements. This assay simplifies the identification of suitable enzymes. Moreover, substrate specificities of a lipase for a certain alcohol or acyl acceptor can also be easily studied with this assay.

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.