Physicochemical and kinetic properties of collagenase, native and adsorbed on chitosan

Abstract

One of the modern research areas in biotechnology and medicine is the development of highly stable biopreparations based on immobilized enzymes. Using the method of sorption immobilization, heterogeneous biocatalysts were obtained based on collagenase (EC 3.4.24.3) from Clostridium histolyticum and from medium molecular weight (200 kDa) and high molecular weight (350 kDa) chitosans. The goal of the work was to study the physicochemical and kinetic properties of free and immobilized collagenase from Clostridium histolyticum on a chitosan matrix. The physicochemical and kinetic properties of the enzyme were studied in soluble and immobilized states using classical biophysical and biochemical methods. It was shown that the maximum activity of native collagenase was observed at 37ºС, while in case of the enzyme immobilized on medium and high molecular weight chitosan it was observed at 40ºС. Sorption immobilization of the biocatalyst on a chitosan matrix allowed increasing its stability during incubation within the temperature range of 40-70°C. After incubation at 80 and 90°C, no significant differences in the activity of soluble and immobilized collagenase were found. When studying the dependence of the protease activity of the enzyme on the medium acidity in the pH range from 5.0 to 9.0, it was established that the maximum catalytic ability of soluble collagenase was observed at a pH value of 7.5. The biocatalyst immobilized on medium and high molecular weight chitosans was highly active at pH 6.0-7.5. The catalytic ability of the enzyme in free and in adsorbed states noticeably decreased at pH 8.0, while at pH 9.0 soluble collagenase immobilized on medium and high molecular weight chitosans was completely inactive. The maximum rate of the enzymatic reaction of native and chitosan-immobilized biocatalysts was observed at a substrate concentration of 0.04 mM. Apparent values of V´max и K´m were calculated. The immobilization of collagenase on medium and high molecular weight chitosans did not result in a decrease of the maximum reaction rate or a change in the degree of affinity of the enzyme to the substrate. Using dynamic light scattering, we confirmed that the structure of collagenase in solution was most stable at pH 7.0-8.0, as exactly in this range of [H+] values we discovered the highest content of particles corresponding to the size of native collagenase and the lowest content of its aggregates. The optimal temperature for the best stability value of collagenase (no autolysis processes and minimal particle aggregation) can be considered as being 46°C.