ABSTRACT
A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Fibrin/chemistry , Lipase/analysis , Peptide Hydrolases/analysis , Triglycerides/chemistry , Bacillus/enzymology , Fibrin/metabolism , Lipase/chemistry , Lipase/metabolism , Nephelometry and Turbidimetry , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Rosaniline Dyes , Staphylococcus/enzymology , Triglycerides/metabolismABSTRACT
We have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.
