Isolation and characterization of a phenol degrading Candida albicans TL3 and the study of its Catechol 1,2-dioxygenase

• Main Authors: San-Chin Tsai, 蔡三進
• Other Authors: Yaw-Kuen Li
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/22559315260855921259

博士 === 國立交通大學 === 應用化學研究所 === 95 === A yeast strain isolated from soil was able to utilize phenol as the sole carbon source and was further identified as Candida albicans TL3. This microbe possesses higher tolerance on phenol (24 mM) as well as stronger activity on the rate of phenol degradation than other microorganisms at 30℃. The capability of this strain on phenol degradation is first reported herein. Based on the enzymatic, chromatographic and mass spectrometric analyses, we concluded that C. albicans TL3 follows the ortho-fission pathway on phenol degradation. The optimal activity of phenol hydroxylase and catechol 1,2-dioxygenase were found when this strain grew in culture media containing 22 mM and 10 mM phenol, respectively. In addition to phenol, C. albicans TL3 also exhibited catalytic power on degrading formaldehyde in wastewater directly obtained from phenolic resin-producing factory. The catechol 1,2-dioxygenase (1,2-CTD) induced from Candida albicans TL3 was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and characterized to be a homodimer, with a molecular weight of 32,000 Da for each subunit. The investigation of this eukaryotic 1,2-CTD revealed that the iron content for each subunit, pI value, optimal temperature, and optimal pH are 1 iron/subunit, 5.3~5.7, 25℃ and pH 8.0, respectively. Substrate analysis showed that the purified enzyme belongs to the type I catechol 1, 2-dioxygenase. The study on this eukaryotic 1,2-CTD was reported for the first time. On 2-D gel analysis of the purified 1,2-CTD, five spots with approximately similar molecular weight but with different pIs were found. These spots were further analyzed by MALDI-TOF mass spectrometry. Results suggested that these spots (isotypes) were derived from the same 1,2-CTD. Peptide sequencing on fragments of 1,2-CTD by Edman degradation and MALDI-TOF/TOF analysis provide information of amino acid sequences for BLAST search, the outcome of the BLAST revealed that this eukaryotic 1,2-CTD has high identity with a hypothetical protein, CaO19_12036, from Candida albicans SC5314 (GenBank accession no. XM 717691). We, thus, suggested that the hypothetical protein should be 1,2-CTD.