Proteolytic Activities of Proteinases Aspergillus Ochraceus: Specific Effect on Haemostasis System Proteins

Fungi, especially such microscopic fungus as Aspergillus ochraceus, are known for their ability to synthesize extra-cellular proteolytic enzymes with different substrate specificity, variety of pH- and thermoactivity optimums, which can operate on hominal hemostasis system. This tendency became the basis for the development of laboratory methods for diagnostics of some proteins of coagulative hemostasis, which are important for signification of pathological plights of patients. Some of these methods are based on proteolysis by sensitive chromogenic peptide substrates after pre-incubation with presence of activating proteins. As activators currently used special proteases, eliminated from snakes venom that makes this diagnosticums really expensive. The synthesis of proteolytic enzymes by Aspergillus ochraceus can make this process easier and cheaper.

The aim of this work was to find the activity of proteases synthesized by Aspergillus ochraceus in pH spectrum from 4 to 8.

In this work we used lyophilized preparation from cultural liquid of microscopic fungus Aspergillus ochraceus L-1from the collection of Moscow State University.

It was shown that proteases of this fungus have three proteases with pI 5.05, 6.07 and 6.83. Exactly, proteins with this pI had the maximal plasmin-like  activity. So we decided to take these fractions for analysis of protein C, factor Xactivator, collagenolysis, kaseinolysis, fibrinolysis and fibrinogenolysis activities. We found that plasmin-like activity of proteases was 19.2 unit/ml ×10 -3, 144.5 unit/ml ×10-3 and 32.4 unit/ml ×10-3 accordingly. We have found the correlation between plasmin-like and other types of proteolytic activities/ The values for fibrinolysis was 10.4 unit/ml, 240.9 unit/ml and 39.2 unit/ml for three purifed proteinases; for fibrinogenolysis was 61.1 unit/ml, 171.5 unit/ml and 107.3 unit/ml accordingly. The activating activities  of Factor X and protein C was 3.3 unit/ml ×10-3, 88.9 unit/ml ×10-3 and 21.9 unit/ml ×10-3 and 7.4 unit/ml ×10-3, 165.3 unit/ml ×10-3 and 61.1 unit/ml ×10-3 for isolated proteinases accordingly. In this way, proteinases produced by Aspergillus ochraceus L-1 may have complex effects on haemostasis system proteins: they are able to hydrolyze fibrin and fibrinogen and activate protein C and factor X. These properties are really very perspective for therapeutic and diagnostic medicine for treatment and prevention of thromboembolyc deseases.

In conclusion it is necessary to say that proteases which are synthesized by Aspergillus ochraceus L-1 and can be used in diagnosticums instead of  proteases, eliminated from snakes venom in future.