Purification of the Protease Activator of Protein C of Human Blood Plasma Produced by the Micromycete Aspergillus ochraceus VKM F-4104D

Komarevtsev, S. K.; Popova, E. A.; Kreyer, V. G.; Miroshnikov, Konstantin A.; Osmolovskiy, A. A.

doi:10.1134/s0003683820010093

Cited by 6 publications

(4 citation statements)

References 21 publications

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“…In particular, extracellular protease of A. ochraceus (PAPC-4104) possesses substrate specificity similar to snake venom protein C activators. However, the production of this protein through fungal cultivation results low yields [ 4 , 5 ] and the purification of PAPC-4104 is difficult due to excessive pigment contamination [ 23 ]. Recombinant production of similar fungal alkaline proteases is also hindered by the multidomain nature and complicated translation, folding, and maturation of the target protein [ 35 , 36 , 37 , 38 , 39 ].…”

Section: Discussionmentioning

confidence: 99%

“…To compare the properties of recombinant and native PAPC-4104, the enzyme was isolated and purified from A. ochraceus VKM-F4104D fermentation medium, as described previously [ 23 ]. Briefly, the 500 mL of medium after cultivation of micromycete was filtered through the filter paper for the purpose of removing biomass, and ammonium sulfate was added to the filtrate to 70% saturation.…”

Section: Methodsmentioning

confidence: 99%

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Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma

et al. 2021

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Micromycetes are known to secrete numerous enzymes of biotechnological and medical potential. Fibrinolytic protease-activator of protein C (PAPC) of blood plasma from micromycete Aspergillus ochraceus VKM-F4104D was obtained in recombinant form utilising the bacterial expression system. This enzyme, which belongs to the proteinase-K-like proteases, is similar to the proteases encoded in the genomes of Aspergillus fumigatus ATCC MYA-4609, A. oryzae ATCC 42149 and A. flavus 28. Mature PAPC-4104 is 282 amino acids long, preceded by the 101-amino acid propeptide necessary for proper folding and maturation. The recombinant protease was identical to the native enzyme from micromycete in terms of its biological properties, including an ability to hydrolyse substrates of activated protein C (pGlu-Pro-Arg-pNA) and factor Xa (Z-D-Arg-Gly-Arg-pNA) in conjugant reactions with human blood plasma. Therefore, recombinant PAPC-4104 can potentially be used in medicine, veterinary science, diagnostics, and other applications.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma

et al. 2021

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“…Для получения внеклеточной протеазы продуцент -микромицет A. ochraceus L-1 -культивировали в глубинных условиях в две последовательные стадии, сначала на посевной среде (состав в %: сусло -6,7, глюкоза -1, пептон -0,1; рН 5,5-6,0), а затем -на ферментационной среде (состав в %: глюкоза -3,5, гидролизат рыбной муки -1, NaCl -0,2, крахмал -0,125, пептон -0,1, KH 2 PO 4 -0,05, MgSO 4 -0,05; рН 5,5-6,0), как было предложено ранее [13,14]. Из культуральной жидкости, предварительно отделенной от биомассы фильтрованием через бумажный фильтр, проводили осаждение белков сульфатом аммония (степень насыщения -0,7) с последующим выделением протеазы в соответствии с разработанным ранее способом, включающим этапы гидрофобной (на фенил-сефарозе, GE Healthcare, США) ионообменной (на ДЭАЭ-сефарозе, GE Healthcare, США) и гель-проникающей (на Сефадексе G-50, Pharmacia, Швеция) хроматографии [15]. Сорбенты для хроматографии уравновешивали 50 мМ Трис-HCl-буфером, рН 8,0.…”

Section: материалы и методыunclassified

Comparison of the in Vitro Anticoagulant Action of Proteases Secreted by the Micromycete Aspergillus Ochraceus and Contained in Snake Venom

Osmolovskiy

Dombeck

Hauke

et al. 2023

Vestn. Mosk. Univ. Ser. 16. Biol.

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The in vitro anticoagulant action of proteases secreted by the micromycete Aspergillus ochraceus L-1 and contained in snakes’ venoms (Protac® and RVV-X® preparations) was studied. The severity of the action of micromycete protease in relation to plasmas of humans and warm-blooded animals, as well as human plasmas de cient in certain factors of the hemostasis system, in comparison with snake proteases in reactions with chromogenic peptide substrates of activated protein C and factor X, as well as using the activated partial thromboplastin time (APTT) test. The optimal time of preincubation of the micromycete A. ochraceus L-1 protease with human blood plasma (3 min) and the concentration of the chromogenic peptide substrate of activated protein C (from 0.1 to 0.5 mg/ml) necessary for the correct determination of protein C with her help.

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“…Such methodologies are used either individually or in combinations, accompanied by chromatographic techniques for further purification. Chromatofocusing, fast protein liquid chromatography, high performance liquid chromatography, affinity column chromatography, gel filtration chromatography, ion exchange chromatography and hydrophobic interaction chromatography are commonly employed techniques for fibrinolytic enzyme purification [2,5,[100][101][102][103][104][105][106][107][108]110,[162][163][164][165][166][167][168]. Some recent purification studies of microbial fibrinolytic enzymes employed by researchers are discussed below.…”

Section: Recovery and Purification Of Fibrinolytic Enzymesmentioning

confidence: 99%

Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications

2021

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Cardiac disorders such as acute myocardial infarction, embolism and stroke are primarily attributed to excessive fibrin accumulation in the blood vessels, usually consequential in thrombosis. Numerous methodologies including the use of anti-coagulants, anti-platelet drugs, surgical operations and fibrinolytic enzymes are employed for the dissolution of fibrin clots and hence ameliorate thrombosis. Microbial fibrinolytic enzymes have attracted much more attention in the management of cardiovascular disorders than typical anti-thrombotic strategies because of the undesirable after-effects and high expense of the latter. Fibrinolytic enzymes such as plasminogen activators and plasmin-like proteins hydrolyse thrombi with high efficacy with no significant after-effects and can be cost effectively produced on a large scale with a short generation time. However, the hunt for novel fibrinolytic enzymes necessitates complex purification stages, physiochemical and structural-functional attributes, which provide an insight into their mechanism of action. Besides, strain improvement and molecular technologies such as cloning, overexpression and the construction of genetically modified strains for the enhanced production of fibrinolytic enzymes significantly improve their thrombolytic potential. In addition, the unconventional applicability of some fibrinolytic enzymes paves their way for protein hydrolysis in addition to fibrin/thrombi, blood pressure regulation, anti-microbials, detergent additives for blood stain removal, preventing dental caries, anti-inflammatory and mucolytic expectorant agents. Therefore, this review article encompasses the production, biochemical/structure-function properties, thrombolytic potential and other surplus applications of microbial fibrinolytic enzymes.

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Purification of the Protease Activator of Protein C of Human Blood Plasma Produced by the Micromycete Aspergillus ochraceus VKM F-4104D

Cited by 6 publications

References 21 publications

Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma

Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma

Comparison of the in Vitro Anticoagulant Action of Proteases Secreted by the Micromycete Aspergillus Ochraceus and Contained in Snake Venom

Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications

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