Proteolytic Activity of Ruminal Bacteria: Studies With Butyrivibrio Fibrisolvens (Protease, Anaerobe, Protein)
Cotta, Michael Anthony
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https://hdl.handle.net/2142/71725
Description
Title
Proteolytic Activity of Ruminal Bacteria: Studies With Butyrivibrio Fibrisolvens (Protease, Anaerobe, Protein)
Author(s)
Cotta, Michael Anthony
Issue Date
1985
Department of Study
Animal Science
Discipline
Dairy Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The proteolytic activity of the ruminal anaerobe Butyrivibrio fibrisolvens was examined using azocasein degradation to monitor activity. Growing cells of B. fibrisolvens produced an extracellular proteolytic activity and the rate of enzyme synthesis largely paralleled growth. Protease production was not subject to induction as enzyme activity was always present regardless of the nitrogen source or carbohydrate source provided in the growth medium. Production of proteolytic activity may be subject to repression as ammonia (10 mM) in the presence of peptides (Trypticase, 1 g/l) or high concentrations of peptides (Tryticase, 20 g/l) alone caused approximately a 50% depression in the production of activity. Cultures provided with different carbohydrate sources for growth exhibited differences in protease production and the production of activity was positively correlated with growth rate.
Chemical and physical characteristics of protease activity of mid-logarthmic grown culture were examined. The presence of atmospheric oxygen did not affect activity, but inclusion of cysteine (1 mM) or dithiothreitol (1 mM) in the assay mixture inhibited activity by about 30%. Additions of divalent cations did not stimulate activity. The majority (about 70%) of the extracellular activity could be sedimented by centrifugation (200,000 x g). Maximum enzyme activity occurred at 47(DEGREES)C and between pH 5.5 and pH 7.0. Sulfhydryl protease inhibitors, carboxylprotease inhibitor, and metal chelators had little effect on proteolytic activity. Similarly, trypsin inhibitor, tosyl-1-lysine chloromethyl ketone, and tosylamide phenylethyl chloromethyl ketone were ineffective inhibitors of enzyme activity, but phenylmethyl sulfonyl fluoride caused greater than 90% inhibition of activity.
Attempts to purify protease were unsuccessful. An isolate was obtained from DEAE-Sephadex chromatography that exhibited an estimated molecular weight of 14,000. Samples from ion-exchange chromatography and sediments from ultracentrifugation contained large quantities of carbohydrate in addition to protein.
The data indicate, B. fibrisolvens produces an extracellular proteolytic enzyme(s) that is associated with an extracellular carbohydrate-containing material produced by the organism. The protease activity has characteristics of serine proteases and production of this activity may be under some form of negative control.
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