College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China.
Int J Mol Sci. 2019 Apr 30;20(9):2143. doi: 10.3390/ijms20092143.
DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (Aga1) from . was heterologously expressed in . The purified recombinant Aga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from and , respectively. Aga1 exhibited an optimal temperature and pH of 40 C and 7, respectively. Aga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40C and around 70 C, but was thermolabile at about 50 C. With the agarose as the substrate, the and values for Aga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of Aga1 by more than one fold. In addition, Aga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that Aga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of Aga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of Aga1.
从东南极沿海地区分离到的 DSM 14237 被发现能够水解几种类型的多糖物质。在这项研究中，一种来自 的预测 β-琼脂酶 (Aga1) 在 中异源表达。纯化的重组 Aga1 对琼脂糖、纯琼脂和 、 的粗琼脂分别表现出 29.39、20.20、14.12 和 8.99 U/mg 的比活性。Aga1 的最适温度和 pH 分别为 40°C 和 7。Aga1 在 pH4 至 11 的宽范围内稳定。重组酶表现出异常的热稳定性，即在低于或等于 40°C 和约 70°C 的温度下稳定，但在约 50°C 时不稳定。以琼脂糖为底物，Aga1 的 Km 和 Vmax 值分别为 1.19mg/mL 和 36.21U/mg。还原剂（二硫苏糖醇）使 Aga1 的活性提高了一倍以上。此外，Aga1 耐盐，在 2 M NaCl 存在下保留了约 70%的最大活性。薄层色谱结果表明，Aga1 是一种内切型 β-琼脂酶，能够有效地将琼脂水解为 neoagarotetraose (NA4) 和 neoagarohexaose (NA6)。通过同源建模构建了 Aga1 与 neoagarooctaose (NA8) 复合物的结构模型，解释了 Aga1 的水解模式。
