Gene cloning, phenol-responsive transcriptional profiling and recombinant protein characterization of phenol hydroxylase in Candida tropicalis GY8.

Phenol hydroxylase (PH) plays a central role in phenol biodegradation pathways. In this study, the enzymatic activity of PH in Candida tropicalis GY8 was evaluated following phenol-induced cultivation, and oxidation products were analyzed via high-performance liquid chromatography (HPLC). Two phenol hydroxylase genes, CtPHE1 and CtPHE2, were identified, and their expression patterns were examined using RT-qPCR. Functional analysis of the corresponding proteins was conducted by cloning and expressing the genes in Escherichia coli BL21(DE3). Enzymatic activity reached its maximum after 15 h of cultivation (p < 0.01), and phenol conversion to catechol was verified by HPLC. RT-qPCR results indicated peak transcript levels at 10 h, with CtPHE2 showing consistently higher expression than CtPHE1 at 5, 10, and 15 h (p < 0.01). Both recombinant proteins were capable of phenol degradation, with CtPHE2 exhibiting greater catalytic activity. These findings provide essential molecular information for advancing the understanding of phenol biodegradation mechanisms.