Studies on Production and Purification of Cytochrome c of c Candida krusei and Its Properties

Abstract

A strain of Candida krusei which could produce an exceptionally large amount of cytochrome c was selected from 300 strains of yeast and the fermentation conditions for production of cytochrome c in the cells were studied. Thiamine, glycine and α-ketoglutarate were found from the results to promote the production. By addition of thiamine and glycine to the basal medium, the cells could produce approximately 400 mg of cytochrome c per kg of wet cells (approx. 70% water). It corresponds to about four fold of that produced by the cells of common baker's yeast of Saccharomyces sp. The isolation and purification of cytochrome c from the cells were achieved by means of plasmolysis of cells with ethyl acetate and column chromatography with cation exchangers, and the crystals of cytochrome c were obtained in 58% yield. The α-band maximum was found to be at 549 mμ, which is shorter in wavelength than that of mammalian cytochrome c (550 mμ), and the molar extinction difference between reduced and oxidized forms of this cytochrome c was 24.3 × 10³ M⁻¹ cm⁻¹ (549 mμ), which was approximately 14% higher than that of horse heart cytochrome c. The enzymatic properties of the cytochrome c were examined with a yeast cytochrome oxidase [EC 1. 9. 3. 1], and the results indicated that Candida cytochrome c is oxidized more than ten times faster than horse heart cytochrome c by the oxidase. Other characteristics of this cytochrome c were also described in this paper.