Size-exclusion effect of a substrate upon kinetics of trypsin immobilized on porous bead cellulose. 1. Influence of distribution coefficient of a substrate

A model of heterogeneous biocatalysis, in which kinetics and partition effects are connected via the size-exclusion principle, was worked up experimentally and theoretically. The present paper shows that the maximum relative activity of trypsin (EC 3.4.21.4) immobilized on porous bead (spherical) cellulose is directly proportional to the available distribution coefficient of the substrate. Providing that the excess of substrate is not sufficient (e.g.S/K_m ≈ 1) to safeguard saturated enzyme kinetics, the originally linear relationship of R_a versus K_av turns to an exponential one, without any dependence upon the manner of enzyme immobilization. It is suggested that the above may be a result of partition resistance and that the main factors determining the shape of the R_a versus K_av relation in conditions of substrate shortage are the size and geometry of the matrix. The physical characteristics of the porous carrier as well as the manner of covalent immobilization of the enzyme are all reflected in the constants applied in the derived equations.