School of Medicine and Pharmacy, Key Laboratory of Marine Drugs, Chinese Ministry of Education, Key Laboratory of Glycoscience & Glycotechnology of Shandong Province, Ocean University of China, Qingdao, 266003, China.
Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Appl Microbiol Biotechnol. 2018 Mar;102(5):2203-2212. doi: 10.1007/s00253-018-8762-6. Epub 2018 Jan 20.
It has been a long time since the first α-agarase was discovered. However, only two α-agarases have been cloned and partially characterized so far and the study of α-agarases has lagged far behind that of β-agarases. Here, we report an α-agarase, AgaD, cloned from marine bacterium Thalassomonas sp. LD5. Its cDNA consists of 4401 bp, encoding a protein of 1466 amino acids. Based on amino acid similarity, AgaD is classified into glycoside hydrolase (GH) family GH96. The recombinant enzyme gave a molecular weight of about 180 kDa on SDS-PAGE and 360 kDa on Native-PAGE indicating it acted as a dimer. However, the recombinant enzyme is labile and easy to be fractured into series of small active fragments, of which the smallest one is about 70 kDa, matching the size of catalytic module. The enzyme has maximal activity at 35 °C and pH 7.4, and shows a strong dependence on the presence of calcium ions. AgaD degrades agarose to yield agarotetraose as the predominate end product. However, the hydrolysates are rapidly degraded to odd-numbered oligosaccharides under strong alkaline condition. The spectra of ESI-MS and H-NMR proved that the main hydrolysate agarotetraose is degraded into neoagarotriose, bearing the sequence of G-A-G (G, D-galactose; A, 3,6-anhydro-α-L-galactose). Unlike the alkaline condition, the hydrolysates are further hydrolyzed into smaller degree polymerization (DP) of agaro-oligosaccharides (AOS) in dilute strong acid. Therefore, this study provides more insights into the properties for both the α-agarases and the AOS.
自从发现第一种α-琼脂酶以来，已经过去了很长时间。然而，迄今为止，仅克隆并部分表征了两种α-琼脂酶，其研究远远落后于β-琼脂酶。在这里，我们报道了一种从海洋细菌 Thalassomonas sp. LD5 克隆的α-琼脂酶 AgaD。其 cDNA 由 4401 bp 组成，编码 1466 个氨基酸的蛋白质。根据氨基酸相似性，AgaD 被归类为糖苷水解酶 (GH)家族 GH96。重组酶在 SDS-PAGE 上的分子量约为 180 kDa，在 Native-PAGE 上的分子量为 360 kDa，表明其为二聚体。然而，重组酶不稳定，容易断裂成一系列小的活性片段，其中最小的片段约为 70 kDa，与催化模块的大小相匹配。该酶在 35°C 和 pH 7.4 下具有最大活性，并表现出对钙离子存在的强烈依赖性。AgaD 将琼脂糖降解为主要终产物琼脂四糖。然而，在强碱性条件下，水解产物迅速降解为奇数寡糖。ESI-MS 和 H-NMR 的图谱证明，主要的水解产物琼脂四糖降解为 neoagarotriose，具有 G-A-G（G，D-半乳糖；A，3,6-脱水-α-L-半乳糖）的序列。与碱性条件不同，在稀强酸中，水解产物进一步水解成聚合度（DP）较小的琼脂寡糖（AOS）。因此，本研究为α-琼脂酶和 AOS 的性质提供了更多的见解。
