College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China.
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Mar Drugs. 2019 Sep 23;17(10):545. doi: 10.3390/md17100545.
Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0-10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.
裂合酶是一种重要的酶，用于制备具有特殊生物活性的褐藻胶寡糖（AOS），广泛应用于医药、食品、化工等领域。本研究筛选并鉴定了一种新型的属于 PL7 家族的多功能褐藻胶裂合酶（AlgH）。AlgH 在 45°C 和 pH 10.0 时表现出最高活性，是一种在 pH 6.0-10.0 稳定的碱性酶。该酶对金属离子没有显著依赖性，在高浓度 NaCl 下仍保持不变的活性。为了确定多结构域酶中非催化结构域的功能，构建并鉴定了仅含有催化结构域的重组 AlgH-I 和含有催化结构域和碳水化合物结合模块（CBM）域的重组 AlgH-II。结果表明，通过删除 F5/8 型 C 结构域，可显著提高重组酶的活性和热稳定性。另一方面，酶的底物特异性和作用方式没有改变。褐藻胶可被全长酶和修饰酶完全降解，主要末端产物为褐藻胶二糖、三糖和四糖。由于其热稳定性、pH 稳定性、耐盐性和多功能性，修饰后的褐藻胶裂合酶是一种可用于 AOS 工业生产的稳健酶。
