Isolation and enzyme determination of Candida tropicalis mutants for DCA production

Techniques, named two-step enrichment and double-time replica-plating metho d (TEDR), are described that allow a mutated population of Candida tropical is to be enriched efficiently for mutants deficient in the alkane degradati on pathway (Alk(-)) and to be selected easily for mutants increasing in the DCA (dicarboxylic acids) excretion pathway. After C. tropicalis was mutate d with ethyl methane sulphonate and ultraviolet, the Alk(-) mutants were en riched (the first step enrichment, up to eightfold in one round of enrichme nt) by treatment with nystatin in medium SEL1-1. The mutagen-treated cells were then cultured in medium YPD containing chlorpromazine for further enri ching (the second-step enrichment, up to threefold in one round) the mutant s with an increasing capacity of alpha- and omega -oxidation. On the other hand, the Alk(-) mutants were readily isolated by the SEL1 replica-plating method by using alkane or glucose as the sole carbon source. A total of 43 Alk(-) mutants were isolated from 2 x 10(8) mutagen-treated cells, In the f ollowing steps, by using SELF replica plating, the screening studies showed that of the 43 Alk(-) mutants, 11 strains could accumulate DCA greatly fro m alkane, and strains 1-12 and 1-3, especially, could produce nearly three times as much DCA as the wild-type organism could. The results showed that the strains had more cytochrome P450 activity and a higher converting capac ity of alkane.