The water-dependence of the catalytic activity of bilirubin oxidase suspensions in low-water systems.
We investigated the enzymic activity of bilirubin oxidase when it is suspended as a lyophilized powder in a low-water system. The enzyme required buffer salts and a source of water to show activity. This study investigated the complete range of water thermodynamic activity (a(w)) by combining the use of salt hydrates and two-phase systems with concentrated solutes in the aqueous phase. When free water was added, activity reached a maximum at a defined water content, but this maximum increased with buffer content, suggesting that there was competition for water with the buffer salts from which the enzyme was lyophilized. Alternatively, a range of salt hydrates was used, each able to fix the water activity (a(w)) at a different value. By providing water to the organic solvent phase in this way, the dependency of enzyme activity upon a(w) was investigated and shown to be independent of buffer concentration. However, the optimum a(w) was uncertain because the available a(w) range for salt hydrates is < or = 0.90. Investigation of the remaining water activity range was made possible by using an a(w) depressor (sorbitol) to lower the a(w) of a two-phase system. The optimum a(w) for the bilirubin oxidase activity in this two-phase system was a(w) = 0.936, independent of buffer concentration. The study therefore confirmed the need to control the water 'available' to low-water systems and the dependence of enzyme activity on water thermodynamic activity (a(w)) not water content.
Alston
Mark J
MJ
Research School of Biosciences, Biological Laboratory, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom.
Freedman
Robert B
RB
D001709:Biotechnology; D002021:Buffers; D002384:Catalysis; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013816:Thermodynamics; D014867:Water / Q000737:chemistry* / Q000378:metabolism
D002021:Buffers; D014867:Water; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase