A Collagenolytic Enzyme from Aspergillus oryzae

Abstract

1.A highly purified protease has been prepared from culture filtrates of Aspergillus oqzae. 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.2 . The molecular weight was found to be about 20,000 by elution from a Sephadex 0-75 column, by ultracenttrifugation 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 Cuzf, Hg2+ and Zn2+ ions. It is also inhibited by di-isopropyl phosphofluoridate and other compounds that react specifically with serine.3. The protease splits denatured proteins preferentially, but not solely, a t 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. I n addition, the prot,ease described here is capable of digesting bonds of the polar regions of the native collagen molecule. The protease also acts as an esterase. 4.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.

Comparative electrophoresis and some properties of alkaline proteinases produced by Monascus spp.

“…Moreover, lyophilization did not much affect the enzyme activity. These results were similar to those obtained with the alkaline proteinases detected in Aspergillus and Penicillium (1,3,5,7,11,13,14,16,18). The results obtained here will provide useful information for separating the enzymes.…”

“…Several strains of Aspergillus and Penicillium which are close relatives of Monascus are known to produce the serine-alkaline proteinases described above. Studies of the substrate specificities of some of them indicated that their specificities were quite different from chymotrypsin or trypsin (13,18). Since alkaline proteinases of Monascus were also considered to be serine proteinases, it will also be a subject of considerable interest to characterize them enzymologically and compare them with those of other origins.…”

Collagenolytic Enzymes

“…In all probability, it is a t this stage that the enzymes frequently encountered in animal tissues and capable of cleaving Z-GPGGPA, PZ-PLGPA, and other model pept,ides (see Section III.B.9) play their role in collagen metabolism. They may be peptidases in nature, even quite specific peptidases, but the same type of reaction can also be catalyzed by endopeptidases of poor specificity requirements (180).…”

The Promise of Enzymes in Therapy of Uremia. II. Theoretical Basis – Enzyme Properties

“…The microorganisms synthesizing alkaline proteases include Aspergillus orycae [38,39], Aspergillus flavus [39], Streptococcus griseus [40] and Streptococcus faradiae [41]. Neutral proteases are synthesized, e.g., by Pseudomonas aeruginosa [38]. Mycobacter produces acid proteases [42].…”