Biomass-degrading enzymes can aid the development of new technologies to increase the production efficiency of green fuels like ethanol. This study reports the identification and characterization of a novel glycoside hydrolase family 9 (GH9) member from the digestive gland of the snail Achatina fulica, an endo-β-1,4-glucanase named AfEG66. The enzyme has a high level of identity with other mollusk endoglucanases and harbors a family 2 carbohydrate-binding module (CBM2) linked to a GH9 domain. Recombinant AfEG66 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography followed by ion exchange chromatography. The enzyme was active against barley β-glucan, lichenan, and showed a specific activity of 41.56 ± 1.21 IU/mg towards carboxymethyl cellulose. AfEG66 showed higher activities at pH 6.0–6.5 and 45 °C, being stable at temperatures equal or below 37 °C. The enzyme activity on cello-oligosaccharides with different glucose units was evaluated by thin-layer chromatography and showed the hydrolysis of substrates containing at least three glucose residues. A structural model was obtained to perform docking assays that clarified the hydrolytic properties of this enzyme. The results presented here show that A. fulica has an endogenous cellulase gene that encodes a GH9 endoglucanase associated to a putative CBM2 with β-1,4 glycoside hydrolase activity.

生物质降解酶可以帮助开发新技术，以提高乙醇等绿色燃料的生产效率。本研究报告了来自蜗牛Achatina fulica消化腺的新型糖苷水解酶家族 9 (GH9) 成员的鉴定和表征，这是一种名为 AfEG66 的内切-β-1,4-葡聚糖酶。该酶与其他软体动物内切葡聚糖酶具有高度的同一性，并含有与 GH9 结构域相连的家族 2 碳水化合物结合模块 (CBM2)。重组 AfEG66 在大肠杆菌中表达并通过固定化金属离子亲和层析和离子交换层析纯化。该酶对大麦 β-葡聚糖、地衣多糖有活性，对羧甲基纤维素的比活性为 41.56 ± 1.21 IU/mg。AfEG66 在 pH 6.0-6.5 和 45°C 下表现出更高的活性，在等于或低于 37°C 的温度下稳定。通过薄层色谱法评估了对具有不同葡萄糖单元的纤维寡糖的酶活性，并显示了含有至少三个葡萄糖残基的底物的水解。获得结构模型以进行对接分析，阐明该酶的水解特性。此处显示的结果表明A. fulica 具有内源性纤维素酶基因，该基因编码与具有 β-1,4 糖苷水解酶活性的推定 CBM2 相关的 GH9 内切葡聚糖酶。