Li Jiajing, Xue Changhu, Shen Jingjing, Liu Guanchen, Mei Xuanwei, Sun Menghui, Chang Yaoguang
College of Food Science and Engineering, Ocean University of China, 1299 Sansha Road, Qingdao 266404, China.
J Agric Food Chem. 2024 Jan 17;72(2):1170-1177. doi: 10.1021/acs.jafc.3c06778. Epub 2023 Dec 18.
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.
G-特异性海藻酸裂解酶是海藻酸片段生物降解和寡糖生产的重要工具，在海藻酸精制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-特异性海藻酸裂解酶Aly7Ce，其最佳反应条件为30℃和pH 8.0。通过采用高效体积排阻色谱-可变波长检测器-质谱联用（UPSEC-VWD-MS）方法，证实Aly7Ce是一种随机内切海藻酸裂解酶。其最小底物为四糖，最终产物主要由二糖至四糖组成。采用高效阴离子交换色谱-脉冲安培检测-质谱联用（HPAEC-PAD/MS）方法研究结构不同的不饱和海藻酸寡糖。通过检测以寡糖或多糖为底物的酶促产物中的寡糖模式，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的亚位点特异性表现为-2（M/G）、-1（G）、+1（M/G）和+2（M/G）。Aly7Ce的规则寡糖产物可用于高效制备高纯度的ΔG、ΔGG和ΔGGG。具有明确产物组成和作用模式的G-特异性海藻酸裂解酶Aly7Ce为海藻酸的修饰和结构解析以及寡糖的靶向制备提供了一种新型工具。
