Stability of immobilized α‐chymotrypsin

Abstract

SummaryThe thermal stability of free and immobilized a-chymotrypsin was investigated experimentally and theoretically. The inactivation process of free a-chymotrypsin was analyzed with a kinetic model which included a first-order reaction process and autolysis. The effects of ionic strength, Ca2+ concentration, and temperature are discussed here in terms of the estimated kinetic parameters included in this model. The inactivation process of a-chymotrypsin immobilized onto various supports by several methods was investigated. The contribution of thermal denaturation and autolysis to the inactivation depended upon the method of immobilization. To interpret quantitatively the non-first-order thermal denaturation process of the immobilized enzyme, a model in which the heterogeneity of the immobilized enzyme was taken into account is proposed.

Deactivation kinetics of immobilized α‐chymotrypsin subpopulations

“…While these results must be viewed with some reservation because of the necessity of invoking certain hypotheses in determining these numbers, it is interesting to note that the important trends are the same for both of the hypothetical models represented by eqs. (2) and (3). The activity of enzyme form B, by far the more active enzyme form, remains relatively constant during the initial 60 min of deactivation but then decreases significantly .…”

Development characterization and use of a high‐performance enzymatic time‐temperature integrator for the control of sterilization process' impacts

“…With regard to multiphasic inactivation kinetics of enzymes, few hypothesis have been described and discussed by several authors (Klibanov, 1979;Martinek and Mozhaev, 1985). The first one assumes that multiphasic inactivation curves result from a continuous spectrum of enzyme molecules with different free inactivation enthalpies (Kawamura et al, 1981). The second one assumes a stepwise loss of activity in only one population of immobilized enzymes (Henley and Sadana, 1986).…”

Structure‐function relationships in immobilized chymotrypsin catalysis

“…17 In immobilized enzyme catalysts, heterogeneity in the local structure of the support surface, as well as that of the enzyme's surface, no doubt combine to produce heterogeneity in the orientation, conformation, and extent of attachment (i.e., the number of bonds between enzyme and support) of the immobilized protein. 6,13 Assorted immobilized enzyme states may result, each with its own free energy, activity, and stability, where here stability is defined as the resistance to loss of catalytic activity. For each immobilized enzyme preparation the sum total of bound protein consists of different immobilized enzyme subpopulations, each of which may have a different active site configuration which contributes a characteristic component to the overall EPR spectrum.…”

“…Enhanced stability of enzymes to thermal denaturation upon immobilization has been reported in several cases, [1][2][3][4] and recently efforts have been made to understand further and optimize these effects by comparing the stabilities of enzymes immobilized by different methods to a wide variety of support materials. 5,6 In addition to studies of this nature, there are many articles, primarily theoretical, which describe possible influences of mass transfer of substrates and products on overall reaction rates of enzymes immobilized to solid supports. Graphical methods for extracting the intrinsic kinetic parameters of immobilized enzymes from diffusion-limited data have been outlined, 7,8 as well as solutions to the appropriate diffusion-reaction boundary value problems for various support geometries and reaction kinetics.…”