Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.

中文翻译：

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嗜碱性细菌莱茵芽孢杆菌 G1 内切-β-1,3-葡聚糖酶的功能表征和产物特异性

来自嗜碱细菌莱茵芽孢杆菌 G1 (Blg32) 的内切-β-1,3-葡聚糖酶由 284 个氨基酸组成，预测分子量为 31.6 kDa，在大肠杆菌中表达并纯化至均质。在此，分析了可能参与糖分子生产的 Blg32 特征、底物和产物特异性以及结构特征。该酶在 70 °C 的温度、8.0 的 pH 值下发挥最佳作用，其催化活性被 Mn2+ 强烈增强。值得注意的是，纯化的酶在高温和碱性条件下高度稳定。它对海带多糖的活性最高 (376.73 U/mg)，其次是凝胶多糖和酵母 β-葡聚糖。与不溶性底物（凝胶多糖）相比，Blg32 对可溶性底物（海带多糖）的活性增加了 62%。海带多糖的水解产物是聚合度（DP）为1至5的低聚糖，主要产物是海带三糖（DP3）。这表明 Blg32 的活性位点可以识别多达五个葡萄糖单位。高浓度的 Blg32 主要产生葡萄糖，而低浓度的 Blg32 产生具有不同 DP（主要是 DP3）的寡糖。构建了 Blg32 的理论结构模型，结构分析表明 Trp156 参与多个疏水堆积相互作用。预测氨基酸参与底物识别和结合。还表明 Blg32 的催化槽具有窄角度，从而限制了底物结合反应。