N. S. Egorov

Screening of Producers of Proteinases with Fibrinolytic and Collagenolytic Activities among Micromycetes

Screening for producers of proteinases with fibrinolytic (plasmin-like and plasminogen-activating) and collagenolytic activities was carried out among 83 strains of microscopic fungi belonging to various ecological groups. Entomopathogenic micromycetes secreted proteinases with higher fibrinolytic and collagenolytic activity than saprotrophic, potentially phytopathogenic, and epiphytic strains. Micromycete strains possessing proteolytic enzymes with collagenase activity were revealed, as were the strains producing proteinases with plasminlike activity. None of the strains studied secreted proteinases possessing only plasminogen-activating activity. Tolypocladium inflatum k1 was found to be a producer of extracellular proteinases with high plasminogen-activating, plasminlike, and collagenolytic activities. The specific plasminogen-activating activity of T. inflatum k1 was shown to be 20% higher than its plasminlike activity.

Aspergillus ochraceus Micromycetes - Producers of Extracellular Proteinases - Protein C Activators of Blood Plasma

Natural isolates of Aspergillus ochraceus micromycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus micromycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombinand plasmin-like activities in the growth dynamics of producers.

The effects of micromycete extracellular proteases of the Aspergillus genus on the proteins of the haemostatic system

The micromycetes of the Aspergillus genus were screened for the evaluation of their capacity to secrete extracellular proteolytic enzymes effecting proteins of the human hemostatic system. Aspergillus extracellular proteases were found to cleave chromogenic peptide substrates specific for the hemostatic proteins, as well as to activate some proenzymes (protein C, factor X, and prothrombin). The activation of human blood plasma X-factor by micromycete extracellular proteases was first shown.

Fibrinolytic and collagenolytic activity of extracellular proteinases of the strains of micromycetes Aspergillus ochraceus L-1 and Aspergillus ustus 1

It was shown that extracellular proteinases produced by the strains of micromycetes A. ochraceus L-1 and A. ustus 1 differ by the activity at various pH as well as by the intensity of the effect on fibrillar proteins. It was revealed that the proteinases of A. ochraceus L-1 demonstrated maximum activity during the growth of the producer in the nitrate-free growth medium (the pH of enzyme reaction was 8.0), whereas those of A. ustus 1 showed maximal activity during the growth of the micromycete in the medium containing sodium nitrate (the pH of enzyme reaction was 6.0). Values of specific fibrinolytic and collagenolytic activities of A. ochraceus L-1 were 2.2 and 1.6 times higher than those of A. ustus 1. At the same time, A. ustus 1 showed very low values of total proteolytic (caseinolytic) activity and had a high ratio of fibrinolytic activity to total proteolytic (caseinolytic) activity (6.92). It makes the strain a promising producer of proteinases, which hydrolyze fibrin and collagen.

Production of Extracellular Proteinases - Protein C Activators of Blood Plasma - by the Micromycete Aspergillus ochraceus during Submerged and Solid-State Fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.

Identification of Targets for Extracellular Proteases Activating Proteins of the Haemostatic System Produced by Micromycetes Aspergillus ochraceus and Aspergillus terreus

Differences in the action of extracellular proteases from Aspergillus ochraceus and Aspergillus terreus on proteins of plasma hemostasis which is responsible for the activation initiation of proteins of the prothrombin complex have been revealed. It has been found that proteases produced by A. ochraceus have a direct influence on protein C and factor X while the exoprotease from A. terreus causes their activation indirectly via stimulation of the kallikrein system. The ability of extracellular proteases of micromycetes to activate prekallikrein in human blood plasma has been demonstrated for the first time by the example of A. terreus.

[Properties of extracellular proteinase--an activator of protein C in blood plasma formed by Aspergillus ochraceus].

The properties of an extracellular proteinase activating plasma protein C isolated from the culture supernatant of Aspergillus ochraceus VKM F-4104D have been studied. This enzyme demonstrated a substrate specificity absent of hydrolyzing activity toward chromogenic proteinase substrates. On the basis of inhibitory analysis, the protein C-activating proteinase from A. ochraceus VKM F-4104D appeared to be a serine proteinase-protein C activator, together with that isolated from the venom of Agkistrodon contortrix contortrix. The isolated enzyme was a nonglycosylated protein with a molecular weight of about 33 kDa, pI 6.0 with an observed optimal activity under a pH of 8.0–9.0 and 37°C. A comparison of the properties of the protein C-activating proteinase formed by A. ochraceus and the enzyme derived from the venom of Agk. contortrix contortrix demonstrated a similarity in their properties; however, proteinase from the micromycete appeared to be in the nonglycosylated state and possessed the ability to hydrolyze the chromogenic plasmin substrate H-D-Val-Leu-Lys-pNA.

Production of proteinases with fibrinolytic and fibrinogenolytic activity by a micromycete Aspergillus ochraceus

Fibrinolytic and fibrinogenolytic activity of surface and submerged cultures of a micromycete Aspergillus ochraceus L-1 was studied. Extracellular proteinases produced by A. ochraceus L-1 were found to exhibit specificity against fibrin and fibrinogen and no activity of plasminogen activators. The highest activity was observed in the cultures grown at 28°С and initial pH 7.0. Fibrinolytic activity was shown to be somewhat above 25% of the total plasmin-like activity of A. ochraceus L-1 proteinases.

Ability of extracellular proteinases of micromycetes Aspergillus flavipes, Aspergillus fumigatus, and Aspergillus sydowii to affect proteins of the human haemostatic system

The effect of extracellular proteinases of A. flavipes A17, A. fumigatus D1, and A. sydowii 1 on proteins of the human haemostasis system was studied. It was shown that A. fumigatus D1 proteinases are able to hydrolyze a wide range of chromogenic peptide substrates of specific human proteinases of the haemostatic system. Proteinases formed by A. flavipes A17 and A. sydowii 1 have a narrow specificity, mainly to thrombin and plasmin substrates. It was first shown that proteinase of A. flavipes A17 is capable to activate protein C and Factor X. Extracellular proteinase produced by A. sydowii 1 has greater fibrinolytic activity as compared with proteinases produced by A. flavipes A17 and A. fumigatus D1.

Properties of extracellular plasmin-like proteases of Aspergillus ochraceus micromycete

The properties of two extracellular proteases of Aspergillus ochraceus VKM F-4104D micromycete with plasmin-like activity have been studied. It has been shown that the enzymes differ in pI (5.05 and 6.83) and have similar molecular weights (about 32 and 35 kDa), pH optima (pH 9.0–10.00 at 45°C), and specificities of action on a limited set of chromogenic peptide substrates of trypsin-like proteases. According to inhibitory analysis, both enzymes belong to the serine proteases. Their properties appeared to be similar to those of the protease, protein C activator, which is the main proteolytic enzyme of A. ochraceus VKM F-4104D. Most likely, proteases of this micromyсetes are isoenzymes.