Screening for enzyme activity in turbid suspensions with scattered light

Abstract

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.

Screening of cellulases for biofuel production: Online monitoring of the enzymatic hydrolysis of insoluble cellulose using high‐throughput scattered light detection

“…This assay is based on the BioLector technique, which allows the online monitoring of scattered light intensities in a continuously shaken microtiter plate [28,29]. In preliminary studies, it was shown that the BioLector technique can be applied in general for measuring enzymatic degradation of insoluble substrates [30]. However, this current study focused on proving the applicability of the BioLector technique for assaying, in particular, cellulase adsorption and activity.…”

Directed Evolution: Methodologies and Applications

“…Microtiter plates containing 96 to 9,600 wells have been combined with colorimetric or fluorometric assays to screen for various enzyme properties. In a microtiter plate assay, the protein with its encoding DNA is compartmentalized in a single well; hence, enzymes with different activity profiles are easily distinguished and unable to diffuse or mix with other DNA–protein pairs. − The substrates and products can be easily identified by macroscopic observation or measuring UV/vis absorbance or fluorescence through a plate reader (Figure ). Recently, a microbioreactor system named Biolector has been used to detect protease and cellulase activities. , Biolector enables online monitoring of the light scattering intensity and reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals by adding light excitation and emission filters to the microtiter plate on a specially designed continuous shaking device.…”

“…286 Recently, a microbioreactor system named Biolector has been used to detect protease and cellulase activities. 284,285 Biolector enables online monitoring of the light scattering intensity and reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals by adding light excitation and emission filters to the microtiter plate on a specially designed continuous shaking device. The scattered light signal difference between hydrolysis of an insoluble protein substrate and NADH-coupled enzyme indicates the protease and cellulase activity levels.…”

“…The scattered light signal difference between hydrolysis of an insoluble protein substrate and NADH-coupled enzyme indicates the protease and cellulase activity levels. 284,285 Microtiter plates have also been used as an alternative to shake flasks for growing variants before screening. 287 However, without the help of an automated colony picker and liquid handler, the throughput of the microtiter plate-based method is relatively low with a screening library size of ∼10 4 per experiment.…”

Online monitoring of dissolved oxygen tension in microtiter plates based on infrared fluorescent oxygen-sensitive nanoparticles

“…[ 11 ] have combined the BioLector technology with a liquid handling system (RoboLector) to reduce the experimental efforts and increase the reproducibility. The Bio- and RoboLector platform have become widespread during the last years and is used in numerous biotechnological applications [ 2 , 12 – 15 ]. Besides the scattered light and biogenic fluorescences the dissolved oxygen tension (DOT) of the cultivation broth is another important process parameter.…”