Journal
BIOTECHNOLOGY PROGRESS
Volume 27, Issue 2, Pages 555-561Publisher
WILEY-BLACKWELL
DOI: 10.1002/btpr.519
Keywords
high throughput; protein hydrolysis; microtiter plate; turbid suspension; protease activity
Funding
- Stiftung Industrieforschung, Koln, Germany [S735]
Ask authors/readers for more resources
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,V-app and K-m,K-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. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 555-561, 2011
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available