As the most abundant polysaccharide in brown algae, alginate has become a promising economical material for bioethanol production. Recently, exo-type alginate lyases have received extensive attention because the unsaturated monosaccharides produced by their degradation of alginate can be easily converted into 4-deoxy-l-erythro-5-hexoseulose uronate (DEH), a promising material for bioethanol production and biorefinery systems. In this study, we cloned and characterized an exo-type polysaccharide lyase family 7 (PL7) alginate lyase VxAly7D from the marine bacterium Vibrio xiamenensis QY104. Recombinant VxAly7D was most active at 30 °C and exhibited 21%, 46% and 90% of its highest activity at 0, 10 and 20 °C, respectively. Compared with other exo-type alginate lyases, recombinant VxAly7D was shown to be a bifunctional alginate lyase with higher specific activity towards sodium alginate, polyG and polyM (462.4 ± 0.64, 357.37 ± 0.53 and 441.94 ± 2.46 U/mg, respectively). A total of 13 μg recombinant VxAly7D could convert 3 mg sodium alginate to unsaturated monosaccharides in 1 min with a yield of 37.6%, and the yield reached 95% in 1 h. In addition, the three-dimensional structure of VxAly7D was modelled using the crystal structure of AlyA5 from Zobellia galactanivorans DsijT as the template. The action mode and the end products of the W295A mutant revealed that Trp295 is a key amino acid residue responsible for the exolytic action mode of VxAly7D. Overall, our results show that VxAly7D is a PL7 exo-type alginate lyase with high activity and a high conversion rate at low/moderate temperatures, which provides a useful enzymatic tool for the development of biofuel production from brown algae and enriches the understanding of the structure and functional relationships of polysaccharide lyases.

中文翻译：

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外切型海藻酸裂解酶VxAly7D的生化特性和分子机制及其在制备不饱和单糖中的应用。

作为褐藻中含量最丰富的多糖，海藻酸盐已成为一种很有前途的经济型生物乙醇生产材料。最近，外切型海藻酸裂解酶因其降解海藻酸产生的不饱和单糖可以很容易地转化为 4-deoxy-l-erythro-5-hexoseulose uronate (DEH) 而受到广泛关注，这是一种用于生物乙醇生产和生物精炼的有前途的材料。系统。在这项研究中，我们从海洋细菌厦门弧菌 QY104 中克隆并表征了一种外型多糖裂解酶家族 7 (PL7) 海藻酸裂解酶 VxAly7D。重组 VxAly7D 在 30 °C 时最活跃，分别在 0、10 和 20 °C 时表现出最高活性的 21%、46% 和 90%。与其他外切型海藻酸裂解酶相比，重组 VxAly7D 被证明是一种双功能海藻酸裂解酶，对海藻酸钠、polyG 和 polyM 具有更高的比活性（分别为 462.4 ± 0.64、357.37 ± 0.53 和 441.94 ± 2.46 U/mg）。13 μg重组VxAly7D可在1 min内将3 mg海藻酸钠转化为不饱和单糖，产率为37.6%，1 h内产率达到95%。此外，VxAly7D 的三维结构使用来自 Zobellia galactanivorans DsijT 的 AlyA5 的晶体结构作为模板进行建模。W295A 突变体的作用模式和终产物表明，Trp295 是负责 VxAly7D 的外溶作用模式的关键氨基酸残基。总体而言，我们的结果表明，VxAly7D 是一种 PL7 外切型海藻酸裂解酶，在低/中温度下具有高活性和高转化率，