KINETICS OF CASEIN HYDROLYSIS BY FREE AND IMMOBILIZED PEPTIDASE FROM BACILLUS THURINGIENSIS VAR. ISRAELENSIS IMB B-7465
In the process of proteases immobilization, their conformation, affinity of enzymes to the substrate, as well as other properties, may change. Therefore, their comprehensive study, including the reaction rates, affinity to the substrate and the determination of the mechanism of action using kinetic...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Odessa I. I. Mechnikov National University
2019-05-01
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| Series: | Vìsnik Odesʹkogo Nacìonalʹnogo Unìversitetu: Hìmìâ |
| Subjects: | |
| Online Access: | http://heraldchem.onu.edu.ua/article/view/169214 |
| Summary: | In the process of proteases immobilization, their conformation, affinity of enzymes to the substrate, as well as other properties, may change. Therefore, their comprehensive study, including the reaction rates, affinity to the substrate and the determination of the mechanism of action using kinetic studies of free and immobilized peptidases, is an important task. This work aimed to determine the kinetic parameters of casein hydrolysis catalyzed by free and released from PVA/chitosan films peptidase from B. thuringiensis var. israelensis IMB B-7465. The kinetics of casein hydrolysis of free and released from PVA/chitosan films peptidase from Bacillus thuringiensis var. israelensis IMB B-7465 was studied. It is shown, that only the modified Anson method is applicable for determining the caseinolytic activity of peptidase, released from the matrix. It was revealed, that at relatively low substrate concentrations, the reaction rate of casein hydrolysis catalysed by free and released from PVA/chitosan films peptidase increased proportionally. When substrate concentration increased, the rate value approached its limit, and then began to decrease. That is, in the certain casein concentration range, the enzyme is inhibited by the substrate. The kinetic constants were measured within the ascending part of the curve relating the initial reaction rate and the substrate concentration. It was determined that the entrapment of the enzyme in the PVA/chitosan film does not significantly affect Vmax. of hydrolysis, while Km is increased 1.3-fold. It is associated with decreasing of the affinity of the enzyme to the substrate as a result of conformational changes in the protein globule, or due to the viscosity limitations, caused by polymers of the matrix. Substrate inhibition of free and released from films peptidase was studied. It was shown that the last Kis is 2.3-fold higher, than that of the free enzyme, which allows to hydrolyze higher concentrations of the substrate. |
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| ISSN: | 2304-0947 2414-5963 |