| Abstract
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A procedure was developed for the detection of low molecular mass (<10 kDa) enzymes from microorganisms, commercial enzyme preparations, and mammalian liver tissue samples. Samples were subjected to ultrafiltration with a 10-kDa exclusion membrane and enzyme activities were detected from ultrafiltrates by radial gel diffusion in substrate-laden agar media. Of 257 samples examined, ultrafiltrates from 14 fungal isolates, including Aspergillus niger ATCC 9029, were positive for CMCase and xylanase activities. A purified endoglucanase from Bacillus subtilis PAP 115, with a molecular mass of 30-33 kDa, was also ultrafiltrate-positive for CMCase activity. Denaturing zymogram analyses of the ultrafiltrates from A. niger and B. subtilis revealed that the molecular masses of the enzymes were 23 and 30 kDa, respectively. The ability of the enzymes to pass through the ultrafilters could be attributed to discrepancies in the membrane pore sizes or, more likely, to an elongated, tapered, or compact molecular shape. A denaturing zymogram method was evaluated for its ability to detect endoglucanase components from Trichoderma reesei, Myrothecium verrucaria, and Bacillus subtilis. Enzyme samples from each microorganism were denatured at 95C for 5 minutes in the presence of 3% SDS and were subjected to SDS-polyacrylamide gel electrophoresis. Carboxymethylcellulose was incorporated into the separating gel. After electrophoresis, the gels were washed with water for 1 hour to allow for enzyme renaturation. Endoglucanase activity bands were detected by staining the gels with 0.1% congo red. In general, molecular mass values of endoglucanases identified by zymogram analyses were consistent with molecular mass values of endoglucanases from T. reesei, M. verrucaria, and B. subtilis.
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