Tag Archives: peptidase
Kinetics of casein hydrolysis by peptidase from Bacillus thuringiensis var. israelensis
O. V. Sevastyanov1, Yu. A. Shesterenko1, A. A. Ryzhak1,
I. I. Romanovska1, N. A. Dziubliuk2, L. D. Varbanets2
1A. V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Odesa;
2Danylo Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: romairina@gmail.com
Received: 25 October 2018; Accepted: 14 March 2019
The kinetics of enzyme reaction is generally studied using the Michaelis-Menten equation and various methods of its linearization. Each method has its advantages and drawbacks, so their comparison for determining the kinetics of new enzymes action is topical. The aim of this work was to study the kinetics of casein hydrolysis catalyzed by new peptidase from Bacillus thuringiensis var. israelensis IMB B-7465 using several methods of enzyme activity assessment and Michaelis-Menten equation linearization. The satisfactory agreement between kinetic constants values obtained by the methods of Lineweaver-Burk, Hanes, Eadie-Hofstee, Cornish-Bowden-Eisenthal was established. The Lineweaver-Burk method was shown to be optimal for determining Km and Vmax of casein hydrolysis. Estimation of caseinolytic activity with the use of ortho-phthalic dialdehyde allowed more accurate Vmax determination compared to the use of Anson and Kunitz methods.
Isolation and purification of Bacillus thuringiensis var. israelensis IМV В-7465 peptidase with specificity toward elastin and collagen
N. А. Nidialkova1, L. D. Varbanets1, V. O. Chernyshenko2
1Institute of Microbiology and Virology, National Academy
of Sciences of Ukraine, Kyiv;
e-mail: Nidialkova@gmail.com;
2Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv
Peptidase of Bacillus thuringiensis var. israelensis IМV В-7465 was isolated from culture supernatant using consecutive fractionations by an ammonium sulphate (60% saturation), ion-exchange chromatography and gel-filtration on the TSK-gels Toyoperl HW-55 and DEAE 650(M). Specific elastase (442 U∙mg of protein-1) and collagenase (212.7 U∙mg of protein-1) activities of the purified enzyme preparation were 8.0- and 6.1-fold, respectively higher than ones of the culture supernatant. Peptidase yields were 33.5% for elastase activity and 30.1% for collagenase activity. It was established that the enzyme is serine metal-dependent alkaline peptidase with Mr about 37 kDa. Maximal hydrolysis of elastin and collagen occurs at the optimum pH 8.0 and t° – 40 and 50 °С, respectively. The purified preparation has high stability at pH in the range of 7.0 to 10.0 and 40-50 °С.