Purification and characterization of cellulases from Clostridium papyrosolvens |
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Authors: | Vicenta Garcia,Alejo Madarro,Jose L. Pe a,Francisco Pi aga,Salvador Vall s,Agusti Flors |
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Affiliation: | Vicenta Garcia,Alejo Madarro,Jose L. Peña,Francisco Piñaga,Salvador Vallès,Agusti Flors |
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Abstract: | The extracellular cellulolytic enzyme complex from Clostridium papyrosolvens was isolated from a culture of this organism grown on filter paper. The complex showed xylanase, carboxymethyl cellulase (CMCase) and Avicelase (but not β-glucosidase) activities. Non-denaturing polyacrylamide gel electrophoresis (PAGE) revealed two dominant bands, sited at the origin (corresponding to a fraction of high molecular weight) and at 280 kDa, and eight other very weak bands in the 20–180 kDa MW range. Gel overlay techniques showed strong CMCase activity in the region comprised between the origin and 320 kDa and in three distinct sites in the 40–230 kDa region. Xylanase activities were detected as a continuous band almost covering all the track. SDS—PAGE gave a multiplicity of different intensity bands in the 20–130 kDa range. Cellulolytic activities (CMCase in the 85–90 kDa range) and hemicellulolytic activities (xylanase at approximately 95, 60, 42 and 32 kDa) were detected through application of the corresponding CMC and xylan overlay techniques. Though anion-exchange chromatography (DEAE-Sephadex A-50) and gel filtration (Biogel P-100) techniques did not permit a good separation of the different cellulolytic activities, an 11.3-fold increase of the Avicelase specific activity was achieved in a fraction containing 38.5% of the original total activity. Maximum enzymatic activities in crude preparations were observed at pH 5.4 and 50°C for xylanase and at pH 4.8 and 45°C for CMCase. The Michaelis constants for CMCase and xylanase were respectively: Vmax = 9.1 μg glucose equivalents min? ml?, Km=3.3 g CMcellulose liter? and Vmax=44.5 μg xylose equiuulents min? ml? and Km=2.7 g xylun liter?. Both enzymes were inhibited by a competitive mechanism. |
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Keywords: | Clostridium cellulases purification characterization enzyme kinetics |
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