Oligosaccharide degradation products of alginate (AOS) hold significant potential in diverse fields, including pharmaceuticals, health foods, textiles, and agricultural production. Enzymatic alginate degradation is appealing due to its mild conditions, predictable activity, high yields, and controllability. However, the alginate degradation often results in a complex mixture of oligosaccharides, necessitating costly purification to isolate highly active oligosaccharides with a specific degree of polymerization (DP). Addressing this, our study centers on the alginate lyase AlyB from Vibrio Splendidus OU02, which uniquely breaks down alginate into mono-distributed trisaccharides. This enzyme features a polysaccharide lyase family 7 domain (PL-7) and a CBM32 carbohydrate-binding module connected by a helical structure. Through normal-mode-based docking and all-atom molecular simulations, we demonstrate that AlyB's substrate and product specificities are influenced by the spatial conformation of the catalytic pocket and the flexibility of its structure. The helically attached CBM is pivotal in releasing trisaccharides, which is crucial for avoiding further degradation. This study sheds light on AlyB's specificity and efficiency and contributes to the evolving field of enzyme design for producing targeted oligosaccharides, with significant implications for various bioindustries.

海藻酸寡糖降解产物（AOS）在医药、保健食品、纺织和农业生产等多个领域具有巨大的潜力。酶促藻酸盐降解因其温和的条件、可预测的活性、高产率和可控性而备受关注。然而，藻酸盐降解通常会产生复杂的寡糖混合物，需要昂贵的纯化来分离具有特定聚合度（DP）的高活性寡糖。为了解决这个问题，我们的研究集中在来自灿烂弧菌OU02的藻酸盐裂解酶 AlyB，它独特地将藻酸盐分解为单分布的三糖。该酶具有多糖裂解酶家族 7 结构域 (PL-7) 和通过螺旋结构连接的 CBM32 碳水化合物结合模块。通过基于正态模式的对接和全原子分子模拟，我们证明了 AlyB 的底物和产物特异性受到催化袋的空间构象及其结构灵活性的影响。螺旋状附着的 CBM 在释放三糖方面至关重要，这对于避免进一步降解至关重要。这项研究揭示了 AlyB 的特异性和效率，并为生产目标寡糖的酶设计领域的发展做出了贡献，对各种生物工业具有重大影响。