Department of Life Sciences, Graduate School of Incheon National University, Incheon, 22102, Republic of Korea.
Department of Food Science and Technology, Chungnam National University, Daejeon, 34134, Republic of Korea.
J Microbiol. 2022 Apr;60(4):375-386. doi: 10.1007/s12275-022-1507-3. Epub 2022 Feb 14.
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 Cu, Hg, and Zn, while it was slightly enhanced by Co, Ca, Ni, and Fe. 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.
创伤弧菌 MO6-24/O 有三个被注释为支链酶或普鲁兰酶基因的基因。其中，由 VVMO6_03032（vvde1）基因编码的蛋白在氨基酸序列水平上与枯草芽孢杆菌的糖原分支酶 AmyX（40.5%）和大肠杆菌的 GlgX（55.5%）的相似度更高，而与其他两个基因编码的蛋白相似度较低。vvde1 基因编码的蛋白分子量为 75.56kDa，纯化的 Vvde1 能有效地将糖原和普鲁兰分别水解成短链的麦芽糊精和麦芽三糖（G3）。然而，它对支链淀粉和可溶性淀粉的水解效率较低，对β-环糊精（β-CD）的水解效率则更低。Vvde1 的最适 pH 和温度分别为 6.5 和 25°C。Vvde1 是一种冷适应的支链酶，在 5°C 时仍保持超过 60%的残余活性。它在 25°C 下能稳定保存 2 天，在 35°C 下能稳定保存 1 天，但在 40°C 时急剧失活。Cu、Hg 和 Zn 对 Vvde1 的活性有显著抑制作用，而 Co、Ca、Ni 和 Fe 则对其活性有轻微的促进作用。vvde1 敲除突变体在补充了 1.0%麦芽糊精的培养基中积累的糖原比野生型多；然而，除了 G3 之外，糖原的侧链长度分布与野生型相似，而 G3 在突变体中更为丰富。因此，Vvde1 似乎以聚合度 3（DP3）作为特定的分支长度来支解糖原。vvde1 突变显著降低了病原体对秀丽隐杆线虫的毒力。这些结果表明，Vvde1 可能是一种独特的糖原分支酶，它既参与糖原的利用，又参与糖原分子的形成，并有助于病原体的毒力。
