Identification and properties of two extracellular proteases from Brevundimonas diminuta

Extracellular proteases from Brevundimonas diminuta (syn. Pseudomonas diminuta) were studied in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) containing a copolymerized substrate. Two proteases were detected migrating at 67kDa and 50 kDa: both of them hydrolysed preferentially gelatin, but casein was also degraded and a slight hydrolysis was observed with hemoglobin. No detectable extracellular proteolytic activity was found in bovine serum albumin-containing gels. The optima temperature and pH for proteolytic activity were between 40ºC and 50ºC in a pH ranging from 7.0 to 11.0, respectively. These enzymes were isolated by analytical high performance liquid chromatography (HPLC). Proteases assays with the synthetic substrate Z-Phe-Arg-MCA and inhibitors EGTA, EDTA and 1, 10 phenanthroline point out that these enzymes are metalloproteases.

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 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 od 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 proyease liberation. Our results 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.