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EnglishIn pancreatic islets purification, for cell therapy applications, the major enzymes used are obtained from Clostridium hystoliticum; class I and class II collagenases (Coll-G and Coll-H). In a well defined composition Coll-G/Coll-H together enzymes working on hydrophobic amminoacid, the neutral protease (Dispase) or the thermolysin (Thermostable Neutral Protease), are used in Langerhans islets purification. By electrophoresis and gelatin zymography approaches, in combination to densitometry quantitative valuation we have compared in composition, stability and autodigestion processes C. hystoliticum collagenases, Neutral protease and Thermolysin from two different producers, Roche and Serva. On analyzed enzymes we have, also, explored about their stability when in solution in relation to storage processes at different temperatures (-20°C; 0°C and r.t.) and on their type-I collagen degradation activity when used at different temperatures (working temperatures: 25°C; 30°C; 37°C and 42 °C). Our results revealed an heterogeneous composition of the different blend of collagenases respect the HPLC profiles publish by vendors and an heterogeneity by lot to lot; moreover, we observed contamination from other enzymes in analysed samples. In fact, in gelatin zymographyes several digestive bands were catalytic active showing very high complex degradative patterns. Additionally, neutral protease from Serva contains gelatinolytic activities; not detected in thermolysin Roche. These data strongly suggest that a not controlled digestive processes can be associate to contaminants present in the blends and also due to auto-catalytic processes. In particular, the presence of low molecular weight gelatine/degradative activities denote the islets digestion process can not be control. Generation of recombinant collagenases probably could be the solution to overcome the variability in the extractive processes.
Salamone, M., Seidita, G., Cuttitta, A., Rigogliuso, S., Bertuzzi, F., & Ghersi, G. (2009). EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION. In Atti del VII congresso: Excerpts from DBCS (pp.51-51). Palermo : Università degli Studi di Palermo.
| Autori: | Salamone, M.; Seidita, G.; Cuttitta, A.; Rigogliuso, S.; Bertuzzi, F.; Ghersi, G. |
| Titolo: | EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION |
| Settore Scientifico Disciplinare: | Settore BIO/10 - Biochimica |
| Data di creazione: | 2009-12 |
| Nome del convegno: | Congresso DBCS 2009 |
| Luogo del convegno: | Palermo |
| Anno del convegno: | 17-18 Dicembre 2009 |
| Numero del convegno: | VII |
| Data di pubblicazione: | 2009 |
| Numero di pagine: | 1 |
| URL: | http://www.unipa.it/dipbio/congresso2009/lavori/%20DBCS%202009%20p.%2051.pdf |
| Citazione: | Salamone, M., Seidita, G., Cuttitta, A., Rigogliuso, S., Bertuzzi, F., & Ghersi, G. (2009). EVALUATION OF STABILITY AND ENZYMATIC ACTIVITIES OF PROTEOLYTIC ENZYMES USED IN PANCREATIC ISLET TRANSPLANTATION. In Atti del VII congresso: Excerpts from DBCS (pp.51-51). Palermo : Università degli Studi di Palermo. |
| Tipologia: | 0 - Proceedings (TIPOLOGIA NON ATTIVA) |
| Appare nelle tipologie: | 0 - Proceedings (TIPOLOGIA NON ATTIVA) |
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