Trypsin like activity (TLA) in collagenase containing tissue dissociation enzyme (TDE) products refers to the presence of enzyme activity that can cleave N-benzoyl-L-arginine ethyl ester (BAEE), a substrate designed to detect trypsin activity. For many years it has been known that clostripain was likely responsible for most if not all this activity since it is difficult to separate clostripain from collagenase because of its charge heterogeneity¹. Clostripain is a neutral protease synthesized by Clostridium histolyticum during anaerobic fermentation. It is a calcium dependent, sulfhydryl endoproteinase that has specificity similar to trypsin, preferring arginine over lysine residues whereas trypsin cuts arginine and lysine residues with equal effectiveness². The enzyme is active at neutral pH and maximally active under reducing conditions. TLA is a measure of enzyme activity in the absence of reducing agent. This reflects approximately 10-30% of the total clostripain activity detected under reducing conditions. Recently, this activity has gained renewed interest with the report by Brandhorst, et al who showed Serva/Nordmark purified collagenase containing high levels of TLA gave higher human islet yields when compared to similar lots of the same product that had lower TLA³. Based on these data, the authors modified their product specifications for using Serva-Nordmark’s NB-1 purified collagenase product to include those lots that have higher levels of TLA contaminants. This article will discuss earlier experience of using clostripain as a neutral protease in the first formulation of the Liberase HI Purified Enzyme Blend product. It is written to provide additional information on Brandhorst’s et al observation since it unclear at this time what mechanism accounts for clostripain’s affect on human islet yields.

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