Extracellular complex of chitinolytic enzymes of Clostridium paraputrificum strain J4 separated by membrane ultrafiltration
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Acetylglucosaminidase chemistry isolation & purification metabolism MeSH
- Bacterial Proteins chemistry isolation & purification metabolism MeSH
- Chitin metabolism MeSH
- Chitinases chemistry isolation & purification metabolism MeSH
- Clostridium enzymology MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Molecular Weight MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Ultrafiltration methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Acetylglucosaminidase MeSH
- Bacterial Proteins MeSH
- Chitin MeSH
- Chitinases MeSH
Membrane diafiltration was used for separation of the extracellular complex of chitinolytic enzymes of C. paraputrificum J4 free from contaminants with molar mass higher than 100 kDa and lower than 30 kDa. The enzyme complex containing beta-N-acetylglucosaminidase (NAGase) and six endochitinases was concentrated on a membrane with cut-off 30 kDa. In this retentate, the NAGase/endochitinase specific activity was 13.5/6.5-times higher than in the initial culture filtrate. The proportion (in%) of endochitinases: 23 (90 kDa), 42 (86 kDa), 8 (72 kDa), 16 (68 kDa) and 8 (60 kDa) was calculated from their peak areas (determined by densitometry) in images of zymograms. NAGase (38 kDa) was less active and stable at pH lower than 4 and higher than 8 but it was more temperature-stable than endochitinases, especially at 40-60 degrees C. In contrast to endochitinases, the pH optimum of NAGase activity was shifted by ca. 0.7 pH units to the alkaline region. Extracellular NAGase together with six endochitinases secreted by C. paraputrificum J4 were separated by membrane diafiltration and characterized by molar mass, stability and activity in dependence on pH and temperature. The knowledge of composition of chitinolytic enzymes, their pH and temperature stability is useful for optimization of the separation process.
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