Abstract
Yeast (Saccharomyces cerevisiae NCYC No. 240) will synthesise a cytochrome P-450, with the characteristic spectral peak at 450 nm, in reduced form in complex with carbon monoxide. This drug-metabolising enzyme, cytochrome P-450, microsomally-located in the yeast cell, is produced rapidly during early phase of growth, up to 17 hours in 1% and 2. 5% glucose media. None is produced in 0. 1% glucose medium. The cytochrome P-450 subsequently disappears from the yeast, while the appearance of cytochrome a + a[3] can now be noted. Conversely, the cytochrome P-450 concentration parallels the growth curve in high glucose media (5-20%), where cytochrome a + a[3] formation is repressed, presumably due to the low concentration of intracellular cyclic AMP under these conditions. Maximal levels of cytochrome P-450 are observed therefore at the end of the growth phase (40 hrs of culture) in high glucose media. Rapid disappearance of the enzyme occurs after this, as the level of cytochrome a + a[3] rises with falling glucose concentration in the media. Yeast and derived microsomal fraction containing cytochrome P-450, could achieve the 4-hydroxylation of biphenyl, using NADPH or NADH, without any 2-hydroxylation. The efficiency of the yeast enzyme in 4-hydroxylation was similar to that of the liver enzyme. The increment of drug metabolising activity and P-450 level obtained on disruption of yeast suggests that yeast is as active in the undisrupted state. Of the disruption techniques used the Vibromill technique seems generally to give the best activity retention and P-450 content. The yeast enzyme was less thermostable and less radiation stable especially when the enzymes were in the reduced form. cerevisiae appears to carry out drug metabolism similar to the hepatic enzyme system. The derived microsomal fractions from yeast can carry out various drug metabolism reactions. The most active reactions carried out by yeast cytochrome P-450/P-448 were 4-hydroxylation of biphenyl; N-demethylation of N-ethylmorphine and aminopyrene; O-demethylation of p-nitroanisol; O-deethylation of coumarin; O-deethylation of resorufin and hydroxylation of the carcinogen, benzo(a)pyrene. The production of cytochrome P-450 in brewers yeast,has been extensively studied,and it was possible to solubilize cytochrome P-450 using sodium cholate in the presence of various stabilisers. The solubilised P-450 was less stable to storage and heat. Partial purification of the solubilised cytochrome P-450 has been achieved using gel filtration and ion exchange chromatography. The purified sample had considerably increased stability to storage at 4°C. The yeast cytochrome P-450 has been characterised as P-450/P-448 system similar to that of the cytochrome P-450/ P-448 inducible in mammalian liver by polycyclic hydrocarbons. The solubilised yeast and liver cytochrome P-450 has been successfully immobilised on various supports, and the immobilised preparation has been stabilised further, to thermal denaturation, by glutaraldehyde. We have demonstrated the 4-hydroxylation of biphenyl and 3-hydroxylation of benzo-(a)pyrene with immobilised preparation of cytochrome P-450. It is suggested that production of yeast on a large scale, perhaps by continuous fermentation, followed by disruption together with isolation of cytochrome P-450 in immobilised and stabilised form, would permit its use in removing carcinogens and their highly reactive metabolites from food stuffs, such as smoked and toasted foods and some drinks. The enzyme could also be used to prepare useful metabolites of drug and carcinogens, which would otherwise have been very difficult and expensive to synthesise.