Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Characterization of Monofunctional Catalase from Photosynthetic Bacterium Rhodospirillum rubrum S1

  • Lee, Dong-Heon (Department of Life Science, Cheju National University) ;
  • Oh, Duck-Chul (Department of Life Science, Cheju National University) ;
  • Oh, You-Sung (Department of Life Science, Cheju National University) ;
  • Malinverni, Juliana C. (Biotechnology Center for Agriculture and the Environment, Rutgers-The State University of New Jersey, Cook College Campus) ;
  • Kukor, Jerome J. (Biotechnology Center for Agriculture and the Environment, Rutgers-The State University of New Jersey, Cook College Campus) ;
  • Kahng, Hyung-Yeel (Department of Environmental Education, Sunchon National University)
  • Published : 2007.09.30
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Abstract

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from $20^{\circ}C$ to $60^{\circ}C$C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's $K_m$ value and $V_{max}$ of the catalase for $H_2O_2$ were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of $A_{406}$ to $A_{280}$ for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1 in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.

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