The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of − 1.9 °C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.

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A new serine-carboxyl proteinase, called kumamolisin-ac , was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45 kDa, active over a wide temperature range (5.0 Á708C) and extremely acidic pHs (1.0 Á4.0), showing maximal proteolytic activity at pH 2.0 and 608C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 58C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t 1/2 at 808C, 10 h, pH 2.0) and over a broad range of pH (2.0 Á7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the 'S53' family. From the high identity with kumamolisin and kumamolisin-As , known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the 'S53' family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine a-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.