1. Horse liver alcohol dehydrogenase and an NADH analogue, N6-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor.2. The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40 of that obtained in excess of free coenzyme.3. A molar ratio of 1 : 1 between the amount of bound nucleotide and bound enzyme was sufficient to obtain high activities in the absence of free coenzyme.4. The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5. Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue.6. The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.
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.
Various flavins, FMN, FAD, and acriflavin, were immobilized to Sepharose using several different coupling methods. The only product stable enough to permit extended studies was acriflavin coupled to epoxy-substituted Sepharose. The nonenzymic oxidizing capacity towards NAD(P) H was investigated and a 25% specific activity, compared to that of free acriflavin, was observed. The reduced acriflavin was immediately auto-reoxidized in air and could thus be reused. It was shown that acriflavin-Sepharose preparations function as NAD(P)H oxidizing agents in a number of different dehydrogenase systems including lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), alanine dehydrogenase (alaDH), and glutamate dehydrogenase (GDH). The amount of expensive coenzyme necessary for high product formation of such systems was thereby markedly reduced.
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