State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Yuan Ming Yuan West Road No. 2, Haidian District, Beijing 100193, China.
Mar Drugs. 2024 Oct 2;22(10):453. doi: 10.3390/md22100453.
Here, we report on a bifunctional alginate lyase (Vnalg7) expressed in , which can degrade natural into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture) was 15.81-fold higher than that of the original (228.90 U/mL-culture), following engineering modification. The degradation rate reached 52.75%, and reducing sugar reached 30.30 mg/mL after combining Vnalg7 (200.00 U/mL-culture) and 14% (/) for 6 h. Analysis of the action mode indicated that Vnalg7 could degrade many substrates to produce a variety of unsaturated alginate oligosaccharides (AOSs), and the minimal substrate was tetrasaccharide. Site-directed mutagenesis showed that Glu, Glu, Glu, Arg, His, Lys, and Tyr are essential catalytic sites, while Glu, Glu, and Asp play auxiliary roles. Mechanism analysis revealed the enzymatic degradation pattern of Vnalg7, which mainly recognizes and attacks the third glycosidic linkage from the reducing end of oligosaccharide substrate. Our findings provide a novel alginate lyase tool and a sustainable and commercial production strategy for value-added biomolecules using seaweeds.
在这里，我们报告了一种在 中表达的双功能褐藻胶裂解酶（Vnalg7），它可以在未经预处理的情况下将天然 降解为不饱和古洛糖醛酸二糖和三糖。经过工程改造，Vnalg7 的酶活性（3620.00 U/mL-培养物）比原始 （228.90 U/mL-培养物）高 15.81 倍。当将 Vnalg7（200.00 U/mL-培养物）与 14%（/）结合并在 6 小时内作用时，降解率达到 52.75%，还原糖达到 30.30 mg/mL。作用模式分析表明，Vnalg7 可以降解许多底物产生各种不饱和褐藻胶低聚糖（AOSs），最小的底物是四糖。定点突变表明 Glu、Glu、Glu、Arg、His、Lys 和 Tyr 是必需的催化位点，而 Glu、Glu 和 Asp 起辅助作用。机制分析揭示了 Vnalg7 的酶促降解模式，它主要识别和攻击寡糖底物还原端的第三个糖苷键。我们的研究结果为利用海藻生产具有附加值的生物分子提供了一种新型褐藻胶裂解酶工具和可持续的商业生产策略。
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