Abstract
The work described in this thesis attempts to analyse the accumulation and degradation of cytochrome P-450 in Saccharomyces cerevisiae. The effect of environmental parameters such as oxygen and constituents of the growth medium have been examined here in an attempt to understand the mechanism underlying the accumulation and degradation of this enzyme. The highest level of S. cerevisiae cytochrome P-450 accumulation was recorded with a new strain NCYC 754 obtained from NCYC 240 and first investigated here. Cytochrome P-450 was found to accumulate during growth of S. cerevisiae only at high glucose concentrations under conditions of mitochondrial repression. It was found that in non-growing yeast a 100 ml 8% glucose (w/v) solution would enhance cytochrome P-450 accumulation. Scale-up of this effect in a 5 l bioreactor was attempted. In experiments on the removal of oxygen during the exponential growth of S. cerevisiae there was found to be a decline in cytochrome P-450 accumulation in which case it is suggested that oxygen may be acting as a substrate inducer of yeast cytochrome P-450. Culture shake speed was also used to control oxygen availability. An optimum shake speed was found which allowed the greatest rate of cytochrome P-450 accumulation, it was also found that the same shake speed caused the greatest rate of degradation of the enzyme during stationary phase. It was also discovered that semi-anaerobic conditions caused less degradation than aerobic conditions. The agents chloramphenicol, dinitrophenol and cycloheximide offered less protection against degradation than semi-anaerobic conditions. Ethanol was found to induce cytochrome P-450 in S. cerevisiae under conditions where cytochrome P-450 is not normally detectable. Added alkanols, other than ethanol, cause rapid degradation of cytochrome P-450 in non-growing yeast.