The specificities of various acid proteinases from fungi or yeasts were comparatively determined using synthetic peptides as substrates. Swine pepsin was used in a comparative study. The pepstatin-sensitive proteinases exhibited their specificity against aromatic or hydrophobic amino acid residues on both sides of the splitting point in peptide substrates, and some differences were observed depending upon their source. Some of the enzymes, possessing trypsinogen-activating ability, also showed their specificity against L -lysine residue on the carboxyl side of the splitting point. In comparison, a pepstatin-insensitive enzyme A from Scytalidium showed considerably broad specificity. The hydrolysis of peptides by pepstatin-sensitive enzymes was markedly accelerated by elongating the peptide chain by three amino acid residues on both sides of the splitting point, while the elongation of two residues produced the same effect with the pepstatin-insensitive enzyme. Thus, Z-(Ala)2-Lys-(Ala)3 was most susceptible (↑ shows the bond split) to the former enzymes possessing trypsinogen-activating ability, and Z-Phe-Leu-Ala-Ala for the latter one. Another pepstatin-insensitive enzyme from Scytalidium showed negligible activity on the peptides used in this study.

Comparative specificity of microbial proteinases.

Comparison of the specificities of various neutral proteinases from microorganisms

Publisher Summary The chapter discusses endopeptidases and focuses on their three main types: (1) acid proteases, (2) diisopropylphosphofluoridate (DFP)-sensitive alkaline proteases, and (3) metal chelator-sensitive neutral proteases. The acid proteases that include the Aspergillus saitoi acid protease, Rhizopus chinensis acid protease, and Paecilomyces varioti acid protease are characterized by low pH activity and stability profiles, insensitivity to metal chelating agents, phosphorylating compounds, such as DFP and thiol poisons. These enzymes show limited esterolytic activity and are reminiscent of the animal pepsins and rennins. The most widely distributed group of proteolytic enzymes of both microbial and animal origin is that of the the serine proteases, generally identified by their sensitivity to organophosphorus reagents, such as DFP and isopropylmethylphosphofluoridate. In addition to the three broad classes of endopeptidases, a number of different kinds of proteolytic enzymes are produced by bacteria and molds. Another unique group of proteases, which in some cases shares properties with one of the other groups, is the bacterial cell wall lysing proteases. These enzymes by virtue of the specific bonds cleaved have uniquely difierent characteristics from the other enzymes.

20 Other Bacterial, Mold, and Yeast Proteases

Enzymic degradation of collagen.

In media containing collagen as the nitrogen source, the pathogenic yeast Candida albicans secreted a collagenolytic enzyme. Purification of the enzyme from a culture filtrate was achieved by DEAE-Sephacel chromatography at pH 6.7. The molecular weight was found to be 46,000 by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was at pH 4.2. The pH optimum lay between 3.5 and 4.0, and above pH 6.0, the enzyme underwent alkaline denaturation. The enzyme was heat labile, and a decrease in activity was found above 55 degrees C. The enzyme activity was inhibited by the addition of urea, cysteine, and pepstatin. No other inhibitor among those tested had any effect. The C. albicans enzyme degraded both the native acid-soluble collagen and the insoluble dentinal collagen.

https://iai.asm.org/content/iai/53/2/312.full.pdf

Isolation and characteristics of collagenolytic enzyme produced by Candida albicans.

1
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.

2
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.

3
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.

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.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1967.tb19562.x

W. F. Jahn

Papers

A Collagenolytic Enzyme from Aspergillus oryzae