<br>新型 G 特异性藻酸盐裂解酶的作用模式：通过 HPAEC-PAD/MS 测定亚位点特异性,Journal of Agricultural and Food Chemistry

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新型 G 特异性藻酸盐裂解酶的作用模式：通过 HPAEC-PAD/MS 测定亚位点特异性
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2023-12-18 , DOI: 10.1021/acs.jafc.3c06778
Jiajing Li ₁ , Changhu Xue ₁ , Jingjing Shen ₁ , Guanchen Liu ₁ , Xuanwei Mei ₁ , Menghui Sun ₁ , Yaoguang Chang ₁

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G特异性海藻酸盐裂解酶是海藻酸盐片段生物降解和寡糖生产的重要工具，在海藻酸盐精炼研究中具有巨大潜力。本研究克隆、表达并表征了一种新型G特异性藻酸盐裂解酶Aly7Ce，最佳反应条件为30℃、pH 8.0。通过采用 UPSEC-VWD-MS 方法，Aly7Ce 被确认为随机内切藻酸盐裂解酶。其最小底物是四糖，最终产物主要由二糖至四糖组成。采用 HPAEC-PAD/MS 方法研究结构不同的不饱和藻酸盐低聚糖。通过检测以寡糖或多糖为底物的酶产物中的寡糖模式，揭示了Aly7Ce的底物识别和亚位点特异性。 Aly7Ce 主要攻击寡糖底物非还原端的第二个糖苷键。 Aly7Ce 的亚位点特异性为 -2 (M/G)、– 1 (G)、+ 1 (M/G) 和 +2 (M/G)。 Aly7Ce的常规寡糖产品可用于高效制备高纯度的ΔG、ΔGG和ΔGGG。 G特异性藻酸盐裂解酶Aly7Ce具有明确的产物组成和作用模式，为藻酸盐的修饰和结构阐明以及寡糖的靶向制备提供了新的工具。

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Action Pattern of a Novel G-Specific Alginate Lyase: Determination of Subsite Specificity by HPAEC–PAD/MS

G-specific alginate lyases are important tools for alginate fragment biodegradation and oligosaccharide production, which have great potential in alginate refining research. In this research, a novel G-specific alginate lyase Aly7Ce was cloned, expressed, and characterized, with the optimal reaction conditions at 30 °C and pH 8.0. By employing the UPSEC–VWD–MS method, Aly7Ce was confirmed as a random endoacting alginate lyase. Its minimum substrate was tetrasaccharide, and the final product majorly consisted of disaccharide to tetrasaccharide. HPAEC–PAD/MS method was employed to investigate the structurally different unsaturated alginate oligosaccharides. The substrate recognition and subsite specificity of Aly7Ce were revealed by detecting the oligosaccharide pattern in the enzymatic products with oligosaccharides or polysaccharides as substrates. Aly7Ce mainly attacked the second glycosidic linkage from the nonreducing end of oligosaccharide substrates. The subsite specificity of Aly7Ce was revealed as −2 (M/G), – 1 (G), + 1 (M/G), and +2 (M/G). The regular oligosaccharide products of Aly7Ce could be applied for the efficient preparation of ΔG, ΔGG, and ΔGGG with high purity. The G-specific alginate lyase Aly7Ce with a well-defined product composition and action pattern provided a novel tool for the modification and structural elucidation of alginate, as well as for the targeted preparation of oligosaccharides.

更新日期：2023-12-18

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