Improvement of 2'-Hydroxybiphenyl-2-sulfinate Desulfinase, an Enzyme Involved in the Dibenzothiophene Desulfurization Pathway, from Rhodococcus erythropolis KA2-5-1 by Site-Directed Mutagenesis

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  • OHSHIRO Takashi
    Department of Biotechnology, Tottori University
  • OHKITA Ryo
    Department of Biotechnology, Tottori University
  • TAKIKAWA Takeshi
    Department of Biotechnology, Tottori University
  • MANABE Masanori
    Department of Biotechnology, Tottori University
  • LEE Woo Cheol
    Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo
  • TANOKURA Masaru
    Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo
  • IZUMI Yoshikazu
    Department of Biotechnology, Tottori University

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  • Improvement of 2′-Hydroxybiphenyl-2-sulfinate Desulfinase, an Enzyme Involved in the Dibenzothiophene Desulfurization Pathway, from<i>Rhodococcus erythropolis</i>KA2-5-1 by Site-Directed Mutagenesis

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In the microbial dibenzothiophene desulfurization pathway, 2′-hydroxybiphenyl-2-sulfinate is converted to 2-hydroxybiphenyl and sulfinate by desulfinase (DszB) at the last step, and this reaction is rate-limiting for the whole pathway. The catalytic activity and thermostability of DszB were enhanced by the two amino acid substitutions. Based on information on the 3-D structure of DszB and a comparison of amino acid sequences between DszB and reported thermophilic and thermostable homologs (TdsB and BdsB), two amino acid residues, Tyr63 and Gln65, were selected as targets to mutate and improve DszB. These two residues were replaced by several amino acids, and the promising mutant enzymes were purified and their properties were examined. Among the wild-type and mutant enzymes, Y63F had higher catalytic activity but similar thermostability, and Q65H showed higher thermostability but less catalytic activity and affinity for the substrate. To compensate for these drawbacks, the double mutant enzyme Y63F-Q65H was purified and its properties were investigated. This mutant enzyme showed higher thermostability without loss of catalytic activity or affinity for the substrate. These superior properties of the mutant enzyme have also been confirmed with resting cells harboring the mutant gene.

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