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Rational Design of Catechol-2, 3-dioxygenase for Improving the Enzyme Characteristics

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Abstract

Catechol-2, 3-dioxygenase (C23O) from Pseudomonas sp. CGMCC2953 identified in our laboratory, which is one of the key enzymes responsible for phenanthrene biodegradation, was expected to get better characteristics tolerant to environment for its further application. With the aim of improving the enzyme properties by introducing intermolecular disulfide bonds, X-ray structure of a C23O from Pseudomonas putida MT-2, a highly conserved homologous with the C23O from Pseudomonas sp. CGMCC2953, was directly used to find the potential sites for forming disulfide bonds between two monomers of the target C23O. Two sites, Ala229 and His294, were identified and mutated to cysteine, respectively, by using site mutagenesis. The expected disulfide bond between these two CYS residues was confirmed with both molecular modeling and experimental results. The optimum temperature of the mutated enzyme was widened from 40 to 40∼50 °C. The mutated C23O became more alkalescency stable compared with the wild-type enzyme, e.g., 75% of the maximal enzyme activity retained even under pH 9.5 while 50% residue for the wild-type one. Improvement of thermostability of the mutated C230 with the redesigned disulfide was also confirmed.

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Authors and Affiliations

  1. School of Life Science, Fudan University, Shanghai, 200433, People’s Republic of China

    Jiashi Wei & Baorong Lu

  2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People’s Republic of China

    Ying Zhou & Tao Xu

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  1. Jiashi Wei
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  2. Ying Zhou
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  3. Tao Xu
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  4. Baorong Lu
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Correspondence to Jiashi Wei.

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Wei, J., Zhou, Y., Xu, T. et al. Rational Design of Catechol-2, 3-dioxygenase for Improving the Enzyme Characteristics. Appl Biochem Biotechnol 162, 116–126 (2010). https://doi.org/10.1007/s12010-009-8720-y

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  • DOI: https://doi.org/10.1007/s12010-009-8720-y

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