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United States Department of Agriculture

Agricultural Research Service

Title: Measurement of Protease Production by Trichoderma Spp.

Author
item Mischke, Barbara

Submitted to: Fungal Genetics Newsletter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 7, 1996
Publication Date: N/A

Interpretive Summary: Proteases degrade proteins and are considered important regulators of fungal parasitism. A reliable method of measuring the production of proteases by beneficial fungi is needed in order to predict their usefulness in the biological control of plant pathogenic fungi. Both water-soluble and water-insoluble substrates each with an attached dye molecule were compared for their ability to measure protease production. The results suggest that the best water-soluble substrate is azoalbumin and the best water-insoluble substrate is azocoll, both reliably releasing dye in proportion to enzyme activity. The most reliable reaction conditions were determined by measuring the amount of protease produced by three species of Trichoderma. These data will be used by scientists developing beneficial fungi as biocontrol agents of plant pathogenic fungi as well as others interested in mechanism of host-parasite interactions.

Technical Abstract: Various chromogenic substrates were compared and methods were developed for measuring nonspecific proteases from fungi. The best water-soluble substrate, with dye release proportional to enzyme activity and high reproducibility, was azoalbumin. Substrates insoluble in water were preferable for time-course studies. Physical characteristics of azocoll compelled its use because uniform samples could be pipetted when a batch was continuously stirred. For comparison of Trichoderma strains the optimal pH was 7.0. Addition of calcium or serine protease inhibitors did not affect crude protease activity. The optimized protocol was used to measure specific activity of extracellular proteases of biocontrol strains of Trichoderma spp.

Last Modified: 8/30/2014
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