Stability of bilirubin oxidase in organic-solvent media - a comparative-study on 2 low-water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroform-rich continuous organic phase. The kinetics of the inactivation process were best described by a double-exponential equation. Approximately half of enzymatic activity was lost during a ''fast'' phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the ''fast'' phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme in aqueous solution was found to be extremely slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the ''slow'' phase observed in water-in-oil microemulsions.