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.

Keywords: Bacillus atrophaeus 08, elastolytic and fibrinogenolytic activity, pH and thermooptimum, protease, substrate specificity