1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligasaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-L-galactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligasaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligasaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligasaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6-anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligasaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20°C, respectively. In particular, rAhg943 could maintain enzyme activity at 10°C (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca²⁺ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K_m and V_max of rAhg943 toward neoagarobiose were 9.7 mg/mL and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

中文翻译：

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新型 1,3-α-3,6-脱水-L-半乳糖苷酶 Ahg943 的分子表征，具有来自 Gayadomonas joobiniege G7 的耐寒和耐高盐性。

1,3-α-3,6-脱水-L-半乳糖苷酶（α-新琼脂寡糖水解酶）通过将新琼脂二糖水解成单体、D-半乳糖和 3,6-脱水-L-半乳糖来催化琼脂降解的最后一步，其中对藻类生物质的生物工业应用具有重要意义。Ahg943 来自琼脂糖分解海洋细菌Gayadomonas joobiniege G7，由 423 个氨基酸 (47.96 kDa) 组成，包括 22 个氨基酸的信号肽。发现它与来自Zobellia galactanivorans 的 α-新琼脂糖寡糖水解酶 ZgAhgA 具有 67% 的同一性，但与报道的其他 α-新琼脂寡糖水解酶的同一性较低 (< 40%)。从大肠杆菌中纯化的重组 Ahg943 (rAhg943, 47.89 kDa), 经凝胶过滤色谱估计为单体，使其与其他 α-新琼脂寡糖水解酶截然不同。rAhg943 将新琼脂二糖、新琼脂四糖和新琼脂六糖分别水解为 D-半乳糖、新琼脂三糖和新琼脂五糖，并具有共同产物 3,6-脱水-L-半乳糖，表明它是一种外切作用的 α-新琼脂寡糖水解酶，可释放3,6-脱水-L-半乳糖通过水解来自新琼脂寡糖非还原端的 α-1,3 糖苷键。Ahg943活性的最适pH和温度分别为6.0和20℃。特别是，rAhg943 可以在 10°C 下保持酶活性（最大值的 71%）。恢复了 0.5 mM EDTA 对 rAhg943 活性的完全抑制，甚至显着增强了 Ca ²⁺离子。rAhg943 活性在 0.5 M NaCl 时最大，并保持在 3M NaCl 时最大值的 73% 以上。rAhg943 对新琼脂二糖的K _m和V _{max分别为 9.7 mg/mL 和 250 μM/min (3 U/mg)。}因此，Ahg943是一种独特的α-新低聚糖水解酶，具有耐寒、耐高盐的特点，可能以单体形式存在。