Purification and physicochemical properties of Bacillus licheniformis 249 protease with elastolytic and fibrin(ogen)olitic activity
Background. Proteolytic enzymes with a broad substrate specificity are of considerable interest for potential application in biotechnology and medicine. Proteases with specificity for elastin, fibrinogen and fibrinogen are of particular interest, in particular as antithrombotic agents. Marine microorganisms due to adaptive mechanisms to environment could be a potential producers of enzymes capable of maintaining catalytic activity in a wide range of physicochemical conditions. Objective. The aim of this work was to purify the protease produced by Bacillus licheniformis 249, isolated from the bottom sediments of the Black Sea and to study the physicochemical and catalytic properties as well as substrate specificity of enzyme. Methods. The enzyme was obtained from the supernatant of the bacterial culture liquid by precipitation with ammonium sulfate (90% saturation) with subsequent gel-permeation chromatography on Toyopearl HW-65F and ion-exchange chromatography on Toyopearl DEAE-650M. Results. The purified enzyme had a molecular weight of about 30.0 kDa, maximal activity at 37°C, pH 8,0 and 11,0 and was characterized by high elastolytic, fibrinogenolytic and fibrinolytic activities with a values of 2697.3; 1620 and 1615 U/mg protein, respectively. Electrophoretic analysis of fibrinogen hydrolysis products revealed selective cleavage of the Aα chain, which allows us to attribute the studied enzyme to α-fibrinogenases. Conclusion. The combination of elastolytic and fibrin(ogen)olytic activities, as well as stability in a wide range of physicochemical conditions, indicate the promising potential of this enzyme for use in medicine and various biotechnological processes. Keywords: Bacillus licheniformis, elastolytic and fibrinogenolytic activity, pH and temperature optimum, protease purification, substrate specificity