Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Int J Biol Macromol. 2019 Sep 15;137:973-981. doi: 10.1016/j.ijbiomac.2019.07.054. Epub 2019 Jul 8.
Some specific properties of pullulanases usually have a great deal to do with their specific N-terminal CBMs. This paper focusses on characters of the novel carbohydrate-binding module CBM68 from a thermostable pullulanase PulA. As to reveal its different properties with the common module CBM41/CBM41-X45 (from an acid pullulanase PulB), twelve recombinant pullulanases were constructed and named as Pul-1~Pul-12 respectively. Through comparative analysis of their thermostabilities, pH profiles, and kinetic parameters, some rules have been concluded. Compared with CBM41, CBM68 on the N-terminal of molecules could make pullulanases more thermostable, have higher substrate affinity, and have higher catalytic efficiency. While CBM41 had the advantage on improving the acidic stability of pullulanase comparing with CBM68. X45 could help pullulanases fold properly and has impact to the catalytic domains of pullulanase. The catalytic domain of PulA or PulB played important roles in the optimum pH and catalytic efficiency of pullulanases. Moreover, the optimal combination of CBM41-X45 and the catalytic domain of PulA made Pul-7 have the highest catalytic efficiency (1284.68 mL/mg·s) which was 1.74 times higher than that (737.78 mL/mg·s) of PulA. The characteristics of CBM68, CBM41 and CBM41-X45 revealed in this study would serve as a basis for rational design of pullulanases.
某些普鲁兰酶的特定性质通常与其特定的 N 端 CBM 密切相关。本文主要研究一种新型碳水化合物结合模块 CBM68 的特性，该模块来自一种耐热普鲁兰酶 PulA。为了揭示其与常见模块 CBM41/CBM41-X45（来自酸性普鲁兰酶 PulB）的不同性质，构建了 12 种重组普鲁兰酶，并分别命名为 Pul-1~Pul-12。通过对其热稳定性、pH 曲线和动力学参数的比较分析，得出了一些规律。与 CBM41 相比，分子 N 端的 CBM68 可以使普鲁兰酶更耐热、对底物具有更高的亲和力和更高的催化效率。而 CBM41 具有提高普鲁兰酶酸性稳定性的优势，与 CBM68 相比。X45 有助于普鲁兰酶正确折叠，对普鲁兰酶的催化结构域有影响。PulA 或 PulB 的催化结构域在普鲁兰酶的最适 pH 和催化效率中起重要作用。此外，CBM41-X45 和 PulA 催化结构域的最佳组合使 Pul-7 具有最高的催化效率（1284.68 mL/mg·s），比 PulA 的催化效率（737.78 mL/mg·s）高 1.74 倍。本研究中揭示的 CBM68、CBM41 和 CBM41-X45 的特性将为普鲁兰酶的合理设计提供基础。
