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The supply of heterotrophically growing suspensions of Alcaligenes eutrophus PHB-4 with oxygen formed by the continuous addition of H2O2 in the presence of bovine liver catalase was found to be restricted to well-defined conditions. The catalase-H2O2 system proved to be suitable during the growth at low cell densities equivalent to 2 g dry weight/liter. When under these conditions the oxygen concentration was held constant at 1.8 mg O2/liter, the cells grew for 6-8 hr at a rate almost identical to that observed with conventional aeration. However, aeration with H2O2 for longer durations (10-20 hr) and at higher cell densities (5-20 g dry weight/liter) led invariably to cell damage and retardation of growth. The impairment of growth observed during the oxygen supply by the catalase-H2O2 system was traced back to the formation of gradually increasing steady-state concentrations of H2O2 in the medium. Possible sites of cell damage by H2O2 such as membrane function, excretion and function of siderophores, and synthesis of cell polymers have been studied, and the cytotoxic mechanism of low concentrations of H2O2 was discussed.

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Empirical estimations of H2O2 concentration in a system containing bovine liver catalase and continually supplied with H2O2 were done to evaluate the efficiency of the enzyme to cleave H2O2. It was found that the continuous addition of H2O2 leads to the formation of steady-state concentrations of H2O2 in the medium. At a constant catalase concentration both the level and the duration of the steady state are dependent on the flow rate of H2O2. The increase of the catalase concentration in the medium does not change the steady-state level, it merely leads to the maintenance of the steady state for longer durations. At higher flow rates of H2O2, no steady state could be maintained, even when catalase was present in high excess. The incomplete cleavage of H2O2 by catalase under these conditions is due to the low affinity of catalase toward H2O2 (high K m value, apparent K m = 0.1M H2O2) and to the rapid inactivation of the enzyme during the continuous addition of H2O2.

Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1'-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate and kappa-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by the N-demethylation of aminopyrine, the O-demethylation of p-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30-40% and a 60-70% decrease in Vappmax for the O- and N-demethylations were respectively observed. The Vappmax values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4 degrees C, microsomes entrapped inside kappa-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped inside kappa-carrageenan or Ca-alginate can be used to follow up the continuous metabolization of p-nitroanisole for several hours in a stirred-batch reactor.