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KMID : 0545120070170091460
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 9 p.1460 ~ p.1468
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Cloning and Characterization of Monofunctional Catalase from Photosynthetic Bacterium Rhodospirillum rubrum S1
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Lee Dong-Heon
Oh Duck-Chul
Oh You-Sung
MALINVERNI JULIANA C.
KUKOR JEROME J.
Kang Hyung-Yeel
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Abstract
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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, 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 20oC to 60oC. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme’s Km value and Vmax of the catalase for H2O2 were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of A406 to A280 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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KEYWORD
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Rhodospirillum rubrum, monofunctional catalase, overexpression, enzyme purification
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