V. G. Kreyer

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.

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.

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.

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.

Secretion of Proteinases with Fibrinolytic Activity by Micromycetes of the Genus Aspergillus

Proteolytic activity of extracellular enzymes of 11 strains of different Aspergillus species was studied. Comparison of the enzymatic indices of strains grown on agar medium containing either casein or fibrin allowed the selection of the strain Aspergillus terreus 2 as a promising producer of fibrinolytic proteases. It was found that A. terreus 2 proteinases demonstrated maximum activity at pH 8.0. The highest values of fibrinolytic and total proteolytic activities expressed in UTyr (amount of micromoles of tyrosine released from fibrin or casein for 1 min) were 34.0 and 358.3, respectively. Maximum activities were detected when growing the producer on a medium containing only amine nitrogen sources (fish flour hydrolysate and peptone); however, the amount of extracellular protein and the specific fibrinolytic and total proteolytic activities were greater in the medium containing both mineral and amine nitrogen sources (fish flour hydrolysate and sodium nitrate) than in the medium containing only fish flour hydrolysate and peptone as nitrogen sources.

Combined microbiological approach to screening of producers of proteases with hemostasis system proteins activity among micromycetes

Highlights • Most of micromycetes are produced proteolytic enzymes which can affect human hemostasis proteins.• For determination of such activity special chromogenic peptide substrates are used.• Proteases secretion by micromycetes depends on medium composition.

Possibility of Application of Extracellular Protease of the Micromycetе Aspergillus ochraceus VKM F-4104D for Determination of the Protein C Content in Human Blood Plasma

The protein C activator activity, determined in normal plasma by using A. ochraceus protease is comparable with the activity of a commercial protease analogue from the South American copperhead venom (Protac®). As in the case of Protac®, the A. ochraceus protease can be used for protein C determination in plasma with its reduced content. Comparison of the activator protein C activity of A. ochraceus protease and the commercial analogue showed some excess of the activator activity of the fungal preparation, which may be a promising substitute for the snake activator in diagnostical kits for determining the protein C content in clinical laboratories.

Secretion of Extracellular Proteinases Active against Fibrillar Proteins by Micromycetes

It has been shown that micromycetes Aspergillus ustus 1 and Tolypocladium inflatum k1 secrete proteolytic enzymes that possess high collagenolytic, fibrinolytic, and elastolytic activity. The activity of proteinases hydrolyzing fibrillar proteins, which was determined by the cleavage of azo-collagen, was 122.6 × 10–3EAzc/mL in A. ustus 1 and 69.7 × 10–3EAzc/mL in T. inflatum k1 (EAzc is the amount of azocollagen cleaved in 1 min (μg). The maximum values of activity were observed during submerged cultivation of A. ustus 1 for 4 days and of T. inflatum k1 for 5 days. It has been shown that the maximum of collagenolytic and general proteolytic activity during the cultivation of A. ustus 1 are time-separated, unlike T. inflatum k1, which, presumably, can simplify the procedure for obtaining proteinases active against fibrillar proteins.