EVALUATION OF STABILITY ANDENZYMATIC ACTIVITIES OF PROTEOLYTICENZYMES USED IN PANCREATIC ISLETTRANSPLANTATION

In pancreatic islets purification, for cell therapyapplications, the major enzymes used are obtained fromClostridium hystoliticum; class I and class IIcollagenases (Coll-G and Coll-H). In a well definedcomposition Coll-G/Coll-H together enzymes workingon hydrophobic amminoacid, the neutral protease(Dispase) or the thermolysin (Thermostable NeutralProtease), are used in Langerhans islets purification.By electrophoresis and gelatin zymography approaches,in combination to densitometry quantitative valuationwe have compared in composition, stability and autodigestionprocesses C. hystoliticum collagenases,Neutral protease and Thermolysin from two differentproducers, Roche and Serva. On analyzed enzymes wehave, also, explored about their stability when insolution in relation to storage processes at differenttemperatures (-20°C; 0°C and r.t.) and on their type-Icollagen degradation activity when used at differenttemperatures (working temperatures: 25°C; 30°C; 37°Cand 42 °C).Our results revealed an heterogeneous composition ofthe different blend of collagenases respect the HPLCprofiles publish by vendors and an heterogeneity by lotto lot; moreover, we observed contamination from otherenzymes in analysed samples. In fact, in gelatinzymographyes several digestive bands were catalyticactive showing very high complex degradative patterns.Additionally, neutral protease from Serva containsgelatinolytic activities; not detected in thermolysinRoche.These data strongly suggest that a not controlleddigestive processes can be associate to contaminantspresent in the blends and also due to auto-catalyticprocesses. In particular, the presence of low molecularweight gelatine/degradative activities denote the isletsdigestion process can not be control. Generation ofrecombinant collagenases probably could be thesolution to overcome the variability in the extractiveprocesses.