SUBSTRATE SPECIFICITY OF BACILLUS THURINGIENSIS ІМВ В-7324 SERINE ALKALINE PEPTIDASE

Abstract

Aim. Investigation of substrate specificity of serine alkaline protease from Bacillus thuringiensis ІMV B- 324. Methods. To study the substrate specificity of the proteins there were used: casein, elastin, fibrin, fibrinogen and collagen. Determination of the primary substrate specificity was carried out using synthetic chromogenic substrates.The maximal velocity (Vmax) and Michaelis constant (Km) were determined according to the method of Lineweaver-Burk using the curve of dependence of the reaction rate from substrate concentration in double reciprocal coordinates (1/V–1/[S]). Results. It was demonstrated that enzyme has substrate specificity similar to subtilisin-type proteases that was determined from the ability to hydrolyze specific chromogenic substrate Z-Ala-Ala-Leu-pNa i Z-Gly-Gly-Phe-pNa, and it also exhibited the esterase activity. Kinetic parameters of hydrolysis of the specific elastase substrate N-succinyl-Ala-Ala-Ala-pNa are 0.83 mM (Km) and 20.1 mmol∙ml-1∙min-1 (Vmax). Conclusions. The ability of elastolytic peptidase B. thuringiensis ІMV B- 7324 to hydrolyse a wide range of native fibrillar and globular proteins is caused by its specificity to the hydrophobicamino acid residues – alanine, leucine, phenylalanine. Kinetic parameters of the enzyme are the same to pancreatic elastase. Thus enzyme is perspective for practical applications.

Keywords

• bacillus thuringiensis
• еластаза
• субстратна специфічність
• синтетичні субстрати