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KMID : 0880220220600040375
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Journal of Microbiology
2022 Volume.60 No. 4 p.375 ~ p.386
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Characterization of a cold-adapted debranching enzyme and its role in glycogen metabolism and virulence of Vibrio vulnificus MO6-24/O
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Han Ah-Reum
Kim Hae-Young
Park Jong-Tae
Kim Jung-Wan
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Abstract
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Vibrio vulnificus MO6-24/O has three genes annotated as debranching enzymes or pullulanase genes. Among them, the gene encoded by VVMO6_03032 (vvde1) shares a higher similarity at the amino acid sequence level to the glycogen debranching enzymes, AmyX of Bacillus subtilis (40.5%) and GlgX of Escherichia coli (55.5%), than those encoded by the other two genes. The vvde1 gene encoded a protein with a molecular mass of 75.56 kDa and purified Vvde1 efficiently hydrolyzed glycogen and pullulan to shorter chains of maltodextrin and maltotriose (G3), respectively. However, it hydrolyzed amylopectin and soluble starch far less efficiently, and β-cyclodextrin (β-CD) only rarely. The optimal pH and temperature of Vvde1 was 6.5 and 25°C, respectively. Vvde1 was a cold-adapted debranching enzyme with more than 60% residual activity at 5°C. It could maintain stability for 2 days at 25°C and 1 day at 35°C, but it destabilized drastically at 40°C. The Vvde1 activity was inhibited considerably by Cu2+, Hg2+, and Zn2+, while it was slightly enhanced by Co2+, Ca2+, Ni2+, and Fe2+. The vvde1 knock-out mutant accumulated more glycogen than the wild-type in media supplemented with 1.0% maltodextrin; however, the side chain length distribution of glycogen was similar to that of the wild-type except G3, which was much more abundant in the mutant. Therefore, Vvde1 seemed to debranch glycogen with the degree of polymerization 3 (DP3) as the specific target branch length. Virulence of the pathogen against Caenorhabditis elegans was attenuated significantly by the vvde1 mutation. These results suggest that Vvde1 might be a unique glycogen debranching enzyme that is involved in both glycogen utilization and shaping of glycogen molecules, and contributes toward virulence of the pathogen.
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KEYWORD
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Vibrio vulnificus, glycogen debranching enzyme, side chain length, cold-adapted, virulence
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