On the Regulation of the Activity of Immobilized Enzymes

Abstract

Glucose oxidase, hexokinase, trypsin and urease were entrapped, either separately or together, within polyacrylamide particles, using a bead polymerization technique. The pH optima, for the immobilized enzymes in 5 mM buffer, were displaced compared to those of the enzymes free in solution.Thus the pH optimum for trypsin activity towards benzoyl-L-arginine ethyl ester shifted 1.3 pH unit (to pH 9.6) and the pH optimum for glucose oxidase towards glucose shifted 0.3 p H units to the alkaline side (to pH 6.9). Reversely, urease activity towards urea leading to consumption of protons shifted its optimum 0.4 unit to the acidic side (to pH 5.8).The effect on glucose oxidase activity of pH changes caused by activity of coentrapped trypsin or urease was studied. It was found that glucose oxidase activity in the alkaline region was stimulated during simultaneous trypsin activity, whereas a t pH values below that of the optimum for glucose oxidase inhibition occurred. A reversed effect was measured during urease activity. At pH 8.6, for example, glucose oxidase activity was increased by a factor of three to 75O/, of its activity found a t pH optimum due to trypsin activity. On the other hand, a t pH 6.0, i.e. below the optimum, it increased by a factor of two to about 800/, due to urease activity. Both simultaneous trypsin or urease activity gave distorted "two-peak" pH-activity profiles of glucose oxidase.The effect of protons produced by trypsin on the simultaneous activities of glucose oxidase and hexokinase, both competing for the same substrate, glucose, was studied with a gel containing all three enzymes. It was found that the amount of glucose converted per minute by each enzyme reaction was effected. I n one case, a t pH 8.5 in the absence of trypsin activity, about one seventh of the glucose consumption was due to hexokinase activity. On addition of benzoyl-Larginine ethyl ester, hexokinase activity decreased almost to zero, while glucose oxidase activity was stimulated, resulting in practically all conversion being due to the latter.

Immobilization of glycogen phosphorylase b in its active conformation on matrices substituted with an AMP‐analogue

“…As is seen from Table 1, the specific activities obtained for the various preparations are relatively high and lie in the range of 15-30% compared to free enzyme. It appears that the presence of effector or effector-analogue during coupling protects the enzyme in analogy to earlier observations made during the entrapment of hexokinase in polyacrylamide beads [ 19]. The specific activities of Sepharosebound phosphorylase are equivalent to those found by other authors [20].…”

Immobilised enzymes

“…Since many enzymes produce or utilize protons it is possible that such action could modify other enzyme activities and thus regulate metabolic pathways. A model system containing entrapped hexokinase, glucose oxidase, and trypsin was studied [49]. At an assay pH of 8.6 (the optimum for hexokinase) 15% of added glucose was phosphorylated and the remainder oxidized by glucose oxidase (pH optimum 6.6).…”

Effect of shaking speed on immobilization of cephalosporin C acylase: Correlation between protein distribution and properties of the immobilized enzymes

“…Based on diffusional limitations, two types of protein distribution patterns (heterogeneous or homogeneous) are formed that affect the apparent kinetic properties of immobilized enzymes in different ways 16,18,19 . An intraparticle pH gradient is created during substrate hydrolysis, and may affect not only the catalytic efficiency but also the stability of enzyme 20‐24 . Therefore, changes in the microenvironmental pH during catalysis, and their impact on catalytic performances of immobilized enzymes underscore the importance of considering protein distribution in the design of an efficient carrier‐bound biocatalyst.…”