title: 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.
authors.affiliation: AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Huber
authors.forename: Helene
authors.initials: H
authors.lastname: Wulfhorst
authors.forename: Lukas
authors.initials: L
authors.lastname: Maksym
authors.forename: Regina
authors.initials: R
authors.lastname: Stehr
authors.forename: Martin
authors.initials: M
authors.lastname: Pöhnlein
authors.forename: Gernot
authors.initials: G
authors.lastname: Jäger
authors.forename: Antje C
authors.initials: AC
authors.lastname: Spiess
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
mesh_terms: D004798:Enzymes / Q000378:metabolism; D007700:Kinetics; D008027:Light; D009391:Nephelometry and Turbidimetry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000032:analysis; D012542:Scattering, Radiation; D057057:Serine Proteases / Q000378:metabolism; D012995:Solubility; D013535:Suspensions / Q000737:chemistry*
chemical_list: D004798:Enzymes; D010446:Peptide Fragments; D011506:Proteins; D013535:Suspensions; D010447:Peptide Hydrolases; D057057:Serine Proteases