Li Jianfang, Wang Chunjuan, Hu Die, Yuan Fengjiao, Li Xueqing, Tang Shihan, Wu Minchen
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
J Biosci Bioeng. 2017 Mar;123(3):294-299. doi: 10.1016/j.jbiosc.2016.09.009. Epub 2016 Oct 20.
A family 27 carbohydrate-binding module of a Thermotoga maritima β-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 β-mannanase from Aspergillus usamii (AuMan5A), two fusion β-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS) and rigid peptide R (EAAAK). Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant β-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70°C, identical with that of reAuMan5A, while its thermostability and melting temperature (T) reached 68°C and 74.9°C, being 8.0°C and 8.4°C higher than those of the latter, respectively. Additionally, the K values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its k/K (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mg⋅s from 171.2, 97.6 and 56.0 mL/mg⋅s of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK) linker contributed to its improved thermostability and catalytic efficiency.
通过计算机辅助设计从碳水化合物活性酶数据库中选择了嗜热栖热菌β-甘露聚糖酶（TmCBM27）的一个家族27碳水化合物结合模块，其与甘露五糖的结合自由能最低。为了改善米曲霉糖苷水解酶家族5β-甘露聚糖酶（AuMan5A）的酶学性质，通过将TmCBM27融合到其C末端并连接柔性肽F（GGGGS）和刚性肽R（EAAAK）设计了两种融合β-甘露聚糖酶，AuMan5A-F-M和AuMan5A-R-M。通过重叠PCR理论设计构建了两个融合酶基因Auman5A-F-m和Auman5A-R-m。然后，将Auman5A和两个融合基因在毕赤酵母GS115中表达。三种重组β-甘露聚糖酶reAuMan5A、reAuMan5A-F-M和reAuMan5A-R-M被纯化至同质，比活性分别为230.6、153.3和241.7 U/mg。reAuMan5A-R-M的最适温度为70°C，与reAuMan5A相同，但其热稳定性和解链温度（T）分别达到68°C和74.9°C，比后者分别高8.0°C和8.4°C。此外，reAuMan5A-R-M对刺槐豆胶、魔芋胶和瓜尔豆胶的K值从reAuMan5A的1.7、3.8和4.2 mg/mL显著降至0.9、1.9和2.5 mg/mL，而其k/K（催化效率）值从后者的171.2、97.6和56.0 mL/mg·s分别增至287.8、163.7和84.4 mL/mg·s。这些结果证实，由（EAAAK）接头介导的将TmCBM27融合到AuMan5A的C末端有助于提高其热稳定性和催化效率。
