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首页> 外文期刊>Applied Microbiology >Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04
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Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04

机译：一种新型的降解多糖的海洋细菌Flammoviravia sp的新型Exo型β-琼脂酶的生化特性和底物降解模式。菌株MY04

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Exo-type agarases release disaccharide units (3,6-anhydro-l-galactopyranose-α-1,3-d-galactose) from the agarose chain and, in combination with endo-type agarases, play important roles in the processive degradation of agarose. Several exo-agarases have been identified. However, their substrate-degrading patterns and corresponding mechanisms are still unclear because of a lack of proper technologies for sugar chain analysis. Herein, we report the novel properties of AgaO, a disaccharide-producing agarase identified from the genus Flammeovirga . AgaO is a 705-amino-acid protein that is unique to strain MY04. It shares sequence identities of less than 40% with reported GH50 β-agarases. Recombinant AgaO (rAgaO) yields disaccharides as the sole final product when degrading agarose and associated oligosaccharides. Its smallest substrate is a neoagarotetraose, and its disaccharide/agarose conversion ratio is 0.5. Using fluorescence labeling and two-stage mass spectrometry analysis, we demonstrate that the disaccharide products are neoagarobiose products instead of agarobiose products, as verified by ~(13)C nuclear magnetic resonance spectrum analysis. Therefore, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. Moreover, AgaO produces neoagarobiose products by gradually cleaving the units from the nonreducing end of fluorescently labeled sugar chains, and so our method represents a novel biochemical visualization of the exolytic pattern of an agarase. Various truncated AgaO proteins lost their disaccharide-producing capabilities, indicating a strict structure-function relationship for the whole enzyme. This study provides insights into the novel catalytic mechanism and enzymatic properties of an exo-type β-agarase for the benefit of potential future applications.IMPORTANCE Exo-type agarases can degrade agarose to yield disaccharides almost exclusively, and therefore, they are important tools for disaccharide preparation. However, their enzymatic mechanisms and agarose degradation patterns are still unclear due to the lack of proper technologies for sugar chain analysis. In this study, AgaO was identified as an exo-type agarase of agarose-degrading Flammeovirga bacteria, representing a novel branch of glycoside hydrolase family 50. Using fluorescence labeling, high-performance liquid chromatography, and mass spectrum analysis technologies, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. We also demonstrate that AgaO produces neoagarobiose by gradually cleaving disaccharides from the nonreducing end of fluorescently labeled sugars. This study will benefit future enzyme applications and oligosaccharide studies.
机译：外切型琼脂酶从琼脂糖链上释放出二糖单元（3,6-脱水-1-半乳糖吡喃糖-α-1,3-d-半乳糖），并与内切型琼脂酶结合，在其降解过程中发挥重要作用。琼脂糖已经确定了几个外星像。然而，由于缺乏合适的糖链分析技术，它们的底物降解模式和相应的机制仍不清楚。在这里，我们报告AgaO的新特性，AgaO是从Flammeovirga属中鉴定出的一种生成二糖的琼脂酶。 AgaO是一种MY705特有的705个氨基酸的蛋白质。它与已报道的GH50β-琼脂糖序列的序列同一性低于40％。当降解琼脂糖和相关的寡糖时，重组AgaO（rAgaO）产生二糖作为唯一的最终产品。它的最小底物是新琼脂四糖，其二糖/琼脂糖转化率为0.5。使用荧光标记和两阶段质谱分析，我们证明了二糖产物是新琼脂糖产物而不是琼脂二糖产物，这由〜（13）C核磁共振光谱分析证实。因此，我们提供了一种有用的寡糖测序方法来确定糖苷键的酶切模式。此外，AgaO通过逐渐从荧光标记的糖链的非还原端切割单元来生产新琼脂糖产品，因此我们的方法代表了一种新型的生化可视化的琼脂糖酶解构图。各种截短的AgaO蛋白丧失了其产生二糖的能力，表明整个酶具有严格的结构-功能关系。这项研究提供了对exo型β-琼脂糖酶的新型催化机制和酶学性质的见解，以利于未来的潜在应用。重要事项exo型琼脂糖酶几乎可以唯一地降解琼脂糖以产生二糖，因此，它们是重要的工具二糖制剂。然而，由于缺乏合适的糖链分析技术，它们的酶促机制和琼脂糖降解模式仍然不清楚。在这项研究中，AgaO被鉴定为降解琼脂糖的Flammeovirga细菌的外切型琼脂酶，代表了糖苷水解酶家族50的一个新分支。使用荧光标记，高效液相色谱和质谱分析技术，我们提供了有用的寡糖测序法确定糖苷键的酶切模式。我们还证明了AgaO通过从荧光标记糖的非还原末端逐渐切割二糖来生产新琼脂糖。这项研究将有益于未来的酶应用和寡糖研究。

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