A COLLAGENOLYTIC ENZYME FROM ASPERGILLUS ORYZAE - PURIFICATION AND PROPERTIES

A highly purified protease has been prepared from culture filtrates of Aspergillus oryzae. The process consists of four steps, namely the dialysis of the starting material, precipitation with acetone, separation by carrier‐free electrophoresis and fractionation with acetone. The specific activity of the protease is increased 40 times or more. The product was shown to be a single substance by means of the ultracentrifuge, disc electrophoresis and immuno‐electrophoresis. As far as investigated, it is free of non‐specific activities. The molecular weight was found to be about 20,000 by elution from a Sephadex G‐75 column, by ultracentrifugation and by amino acid analysis. The pH optimum normally lies between 9 and 10. The protease cannot be activated with metal ions; it is, however, inhibited by Cu2+, Hg2+ and Zn2+ ions. It is also inhibited by di‐isopropyl phosphofluoridate and other compounds that react specifically with serine. The protease splits denatured proteins preferentially, but not solely, at the peptide bonds of amino acids with acid side chains as shown by investigations on long‐chain model peptides. It also digests native collagen. The mode of splitting is the same as that by which the collagenase from Clostridium histolyticum splits the apolar regions. However, bonds involving hydroxyproline appear to be resistant to attack by the Aspergillus enzyme. In addition, the protease described here is capable of digesting bonds of the polar regions of the native collagen molecule. The protease also acts as an esterase. On the basis of the various types of proteolytic specificity found for the protease a new concept is put forward for understanding collagen breakdown in vivo and in vitro. Copyright © 1968, Wiley Blackwell. All rights reserved