Tag Archives: protease

Influence of heterometallic Ge(IV) – 3d-metals complexes on microbial rhamnosidase, galactosidase and protease activity

25.06.2025   Uncategorized   No comments

O. V. Gudzenko1*, L. D. Varbanets1, I. I. Seifullina2,
О. E. Martsynko2, O. A. Finik3, K. K. Tsymbaliuk2,3

1Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
2Odesa I. I. Mechnikov National University, Ukraine;
3LLC “Inspectorat Ukraine”, Ukraine;
*e-mail: alena.gudzenko81@gmail.com

Received: 18 March 2025; Revised: 12 May 2025;
Accepted: 11 June 2025; Available on-line: 07 July 2025

In recent years, the attention of researchers has been attracted by coordination compounds of germanium with various bioligands, which may be used both as activator or inhibitor of enzymes. The aim of the work was to investigate the effect of new heterometallic Ge(IV) – 3d-metals complexes with 1-hydro­xyethane-1,1-diphosphonic acid and 1,10-phenanthroline on the activity of purified α-L-rhamnosidases produced by Eupenicillium erubescens, Penicillium tardum, Penicillium restrictum, Cryptococcus albidus, α-galactosidase of P. restrictum and proteases with elastolytic and fibrinogenolytic activity of Bacillus sp. The studied compounds (0,1% concentration) activate α-L-rhamnosidase differently depending on its producer. Thus, the activity of α-L-rhamnosidase of E. erubescens was stimulated with [Co(phen)3]4[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·2СН3СООН·30H2O by 200%, of Penicillium tardum – with [Zn(phen)2(H2O)2]2[Zn(phen)(H2O)4]2[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·18H2O by 200%, of Penicillium restrictum – with [Ni(phen)3]4[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·2СН3СООН·26H2O by 67%, of Cryptococcus albidus – wih [Co(phen)3]4[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·2СН3СООН·30H2O by 40%. The α-galactosidase of Penicillium restrictum was not affected by the investigated compounds. Bacillus sp. IMV B-7883 elastase was activated with [Co(phen)3]4[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·2СН3СООН·30H2O by 70%, but was totally inhibited with [Fe(phen)3]4[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·20H2O. The compounds that showed the greatest 200% stimulating effect on the fibrinogenolytic activity of Bacillus sp. IMV B-7883 were [Fe(phen)3]4[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·20H2O and [Zn(phen)2(H2O)2]2[Zn(phen)(H2O)4]2[Ge6(μ-OH)4(μ-O)2(μ-hedp)6]·18H2O.

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Purification and physico-chemical properties of Bacillus atrophaeus protease with elastolytic and fibrinogenolytic activity

04.12.2024   Uncategorized   No comments

O. V. Gudzenko1*, L. D. Varbanets1, V. O. Chernyshenko2,
Y. M. Stohnii2, A. M. Ostapchuk3, V. O. Ivanytsia3

1Institute of Microbiology and Virology named after D. K. Zabolotny,
National Academy of Sciences of Ukraine, Kyiv;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
3Odesa I.I. Mechnikov National University, Odesa, Ukraine;
*e-mail: alena.gudzenko81@gmail.com

Received: 18 September 2024; Revised: 21 October 2024;
Accepted: 21 November 2024; Available on-line: 17 December 2024

Microbial proteases, among which proteases capable of cleaving elastin, fibrin, fibrinogen, and collagen, have been a matter of interest to researchers due to their significant biotechnological potential along with low production cost. We previously showed that Bacillus atrophaeus 08 synthesizes an extracellular protease complex that exhibits high elastolytic, fibrinogenolytic, fibrinolytic activity, and minor caseinolytic and collagenase activity. The aim of the work was to isolate and purify the Bacillus atrophaeus 08 protease from the culture liquid supernatant and to study the physicochemical properties and substrate specificity of enzyme preparation. Precipitation with ammonium sulfate of 90% saturation, gel-permeation and ion-exchange chromatography were used in the experiment. According to the data obtained, the yield of the purified enzyme with a molecular weight of about 30 kDa was 6%, its elastase activity increased 30 times (420 U/mg protein), and fibrinogenolytic activity 31.8 times (350 U/mg protein). In addition, it also exhibited fibrinolytic (35.3 U/mg protein), minor caseinolytic activity (1.2 U/mg protein) and no collagenase activity. The optimum of elastin hydrolysis was at 37°C, pH 3.0 and 9.0-10.0, the optimum for fibrinogen hydrolysis was 12°C, pH 4.0. SDS-PAAG electrophoresis showed that the Bβ-chain of fibrinogen was almost not cleaved even after 1 h of incubation with the enzyme, while the Aα-chain disappeared already at the 30th min with the production of fragments with M.W. of about 30-45 kDa. The activity of the studied enzyme preparation towards fibrin was much lower than towards fibrinogen.

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Mapping of residues of fibrinogen cleaved by protease II of Bacillus thuringiensis var. israelensis IMV B-7465

19.07.2016   Uncategorized   No comments

E. M. Stohniy1, V. O. Chernyshenko1*, N. A. Nidialkova2, A. V. Rebriev1,
L. D. Varbanets2, V. E. Hadzhynova1, T. M. Chernyshenko1,
I. M. Kolesnikova1, E. V. Lugovskoy1

1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
2Zabolotny Institute of Microbiology and Virology, National Academy
of Sciences of Ukraine, Kyiv;
*e-mail: bio.cherv@gmail.com

The limited proteolysis of macromolecules allows obtaining the fragments that preserve the structure and functional properties of the whole molecule and could be used in the study of proteins structure and function. Proteases targeted to fibrinogen and fibrin are of interest as the tool for obtaining of functionally active fragments of fibrin(ogen) and for the direct defibrination in vivo. That is why the aim of the present work was to study the proteolytic action of Protease II (PII) purified from Bacillus thuringiensis var. israelensis IMV B-7465 on fibrinogen.
Hydrolysis products of fibrinogen by PII were analysed by SDS-PAGE under reducing conditions with further immunoprobing using the mouse monoclonal 1-5A (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibodies. It was shown that PII cleaved preferentially the Aα-chain of fibrinogen splitting off the peptide with apparent molecular weight of 10 kDa that corresponded the C-terminal part of Aα-chain of fibrinogen molecule.
MALDI-TOF analysis of hydrolysis of fibrinogen was performed using a Voyager-DE. Results analyzed by Data Explorer 4.0.0.0 allowed to detect the main peak occurring at mass/charge (M/Z) ratio of 11 441 Da. According to “Peptide Mass Calculator” this peptide corresponded to fragment Аα505-610 of fibrinogen molecule. The result showed that PII cleaves the peptide bond AαAsp-Thr-Ala504-Ser505.
Thus, PII can be used for the obtaining of unique fragments of fibrinogen molecule. As far as αC-domain contains numerous sites of fibrin intermolecular interactions we can consider PII as a prospective agent for their study and for the defibrination.

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