To characterize the hydrolysis product and the substrate binding in the catalytic cavity of α-agarase AgaD. The time course curve showed that AgaD degraded agarose by the endo-type cleavage. AgaD did not degrade agarobiose (A2) and agarotetraose (A4). The minimum-length substrate was agarohexaose (A6), which was cleaved into A2 and A4. Agarooctaose (A8) was cleaved into two molecules of A4. Consistently, TLC and NMR data identified agarotetraose (A4) as the main hydrolysate when agarose was degraded by AgaD. This study confirms AgaD is an endo-type α-agarase and A4 as the main hydrolysis product of AgaD, which suggests the catalytic cavity of AgaD accommodates eight sugar units spanning from − 4 to + 4.

中文翻译：

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α-琼脂酶 AgaD 水解产物和催化腔的表征

表征水解产物和底物结合在 α-琼脂酶 AgaD 的催化腔中。时间过程曲线显示 AgaD 通过内切型裂解降解琼脂糖。AgaD 不降解琼脂二糖 (A2) 和琼脂四糖 (A4)。最小长度的底物是琼脂六糖 (A6)，它被切割成 A2 和 A4。琼脂八糖 (A8) 被切割成两个 A4 分子。一致地，当琼脂糖被 AgaD 降解时，TLC 和 NMR 数据将琼脂四糖 (A4) 鉴定为主要的水解产物。该研究证实 AgaD 是一种内型 α-琼脂酶，A4 是 AgaD 的主要水解产物，这表明 AgaD 的催化腔可容纳从 - 4 到 + 4 的八个糖单元。