Cloning, expression and characterization of 1,2,4-trihydroxybenzene 1,2-dioxygenase from Taiwanofungus camphoratus

• Main Authors: Tzung-Lung Chou, 周宗龍
• Other Authors: Chi-Tsai Lin
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/22024856954651831511

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 101 === Hydroxyquinol 1,2-dioxygenase (HQD) plays an important role in aromatic-compound degradation system. HQD catalyzed hydroxyquinol (1,2,4-trihydroxybenzene, HQ) and produced maleylacetate formation. In this study, a full length cDNA (1,233 bp) encoding a putative HQD from fruiting bodies of Taiwanofungus camphorata was cloned, the open reading frame has 1,035 nucleotides and encodes 344 amino acids. The deduced amino acid sequence is similar to the HQDs from other species. A 3-D structural model of TcHQD was built based on the crystal structure of PpHQD (Psudomonas putida; PDB code 3N9T_A). To further characterize the TcHQD, the coding region was subcloned into an expression vector pYEX-S1 and transformed into Saccharomyces cerevisiae. Subsequently, the purified enzyme showed a single band at molecular mass of approximately 35.0 kDa on 12 % polyacrylamide gel after SDS-PAGE. Furthermore, it was left over 41% at pH 6.0 compaired with pH 7.0 with the enzyme tolerance, the Michaelis constant value (KM) for hydroxyquinol was 36.68 μM, the thermal inactivation of the enzyme showed a half-life of 4.89 min at 58℃ and the inactivation rate constant kd was 1.93×〖10〗^(-3) min-1.