Detection of extracellular proteases from microorganisms on agar plates.

We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

[1]  S. Goldenberg,et al.  Ubiquity of Cysteine‐ and Metalloproteinase Activities in a Wide Range of Trypanosomatids , 1996, The Journal of eukaryotic microbiology.

[2]  M. Rao,et al.  Thermostability of high-activity alkaline protease from Conidiobolus coronatus (NCL 86.8.20) , 1995 .

[3]  P. Brown.  Clinical Trials of a Low Molecular Weight Matrix Metalloproteinase Inhibitor in Cancer , 1994, Annals of the New York Academy of Sciences.

[4]  D. Haas,et al.  Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0. , 1994, FEMS microbiology letters.

[5]  H. Yajima,et al.  Erythromycin inhibits the production of elastase by Pseudomonas aeruginosa without affecting its proliferation in vitro. , 1993, The American review of respiratory disease.

[6]  D. Agard,et al.  Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli , 1992, The Journal of cell biology.

[7]  G. Stewart,et al.  Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects , 1987, Applied and environmental microbiology.

[8]  G. Valdez,et al.  Effect of pH and Temperature on the Proteolytic Activity of Lactic Acid Bacteria , 1985 .

[9]  R. Juliano,et al.  Detection of proteases in polyacrylamide gels containing covalently bound substrates. , 1984, Analytical biochemistry.

[10]  M. Trost,et al.  A comparison of secretory proteinases from different strains of Candida albicans. , 1982, Sabouraudia.

[11]  H. Elwing,et al.  Determination of proteolytic activity: a sensitive and simple assay utilizing substrate adsorbed to a plastic surface and radial diffusion in gel. , 1981, Analytical biochemistry.

[12]  E. Dowdle,et al.  Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. , 1980, Analytical biochemistry.

[13]  H. Juffs.  Effects of temperature and nutrients on proteinase production by Pseudomonas fluorescens and Ps. aeruginosa in broth and milk. , 1976, The Journal of applied bacteriology.

[14]  D. Dabich,et al.  A sensitive polyacrylamide disc gel method for detection of proteinases. , 1974, Analytical biochemistry.

[15]  S. Meyers,et al.  Extracellular proteinases of yeasts and yeastlike fungi. , 1968, Applied microbiology.

[16]  M. C. Geokas,et al.  A new ultrasensitive method for the determination of proteolytic activity. , 1968, Clinica chimica acta; international journal of clinical chemistry.

[17]  M. L. Anson,et al.  THE ESTIMATION OF PEPSIN, TRYPSIN, PAPAIN, AND CATHEPSIN WITH HEMOGLOBIN , 1938, The Journal of general physiology.

[18]  K. Morihara,et al.  Pseudolysin and other pathogen endopeptidases of thermolysin family. , 1995, Methods in enzymology.

[19]  K. Morihara,et al.  Serralysin and related bacterial proteinases. , 1995, Methods in enzymology.

[20]  J. D. Grubb.  Assays for bacterial type I collagenases. , 1994, Methods in enzymology.

[21]  P. Ciborowski,et al.  Zymographic techniques for detection and characterization of microbial proteases. , 1994, Methods in enzymology.

[22]  J. D. Grubb.  Purification and assays of bacterial gelatinases. , 1994, Methods in enzymology.

[23]  B. Iglewski,et al.  [44] Elastase assays , 1994 .

[24]  B. Iglewski,et al.  Elastase assays. , 1994, Methods in enzymology.

[25]  N. Lewis,et al.  A note on nutritional influence on extracellular protease synthesis in Aeromonas hydrophila , 1985 .