title: Overexpression, Purification and Characterization of Thermostable Catechol 2,3-dioxygenase.
abstract: Catechol 2,3-dioxygenase(EC 1.13.11.2) from Bacillus stearothermophilus has been shown to be highly thermostable. In order to obtain sufficient enzyme for its characterization, and to analyze the molecular basis of its intrinsic stability, the gene coding for this enzyme was sub-cloned and overexpressed in E. coli. After heat denaturation, ammonium sulfate fractionation, DEAE-52 ion-exchange chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography, the enzyme was purified to homogeneity and the yield was 16%. The enzyme is a homotetramer. The molecular weight of each subunit is 36.4 kD as determined by SDS-PAGE. Using the tyrosine difference spectrum method, the extinction coefficient of the enzyme at 279.2 nm is estimated as 89(mmol/L)(-1).cm(-1), the optimum temperature and pH of the enzyme is 60 degrees and 8.0, respectively. The K(m) for catechol of the enzyme is 1.24x10(-5) mol/L, K(cat) is 24 000 min(-1). Acetone competitively inhibited the enzyme activity to catechol, with an inhibition constant K(i) of about 165 mmol/L. The thermostability of the enzyme is reflected by its unaltered catalytic activity over 1 h at 65 degrees. Irreversible thermal denaturation becomes significant between 70-80 degrees. THE pH stability of the enzyme and its resistance toward chemical denaturation such as urea and SDS confirm the intrinsic stability of the enzyme. It was also studied that the effect of different pHs on the molecular structure of the enzyme, using circular dichroism spectra and intrinsic fluorescence analysis.
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai 200433, China. ymmao@fudan.edu.cn
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Chang-Chuan
authors.initials: CC
authors.lastname: Yin
authors.forename: Zuo-Hua
authors.initials: ZH
authors.lastname: Zheng
authors.forename: Zai-Long
authors.initials: ZL
authors.lastname: Cai
authors.forename: Qing
authors.initials: Q
authors.lastname: Xia
authors.forename: Yu-Min
authors.initials: YM
authors.lastname: Mao