Li Xue-Qing, Wu Qin, Hu Die, Wang Rui, Liu Yan, Wu Min-Chen, Li Jian-Fang
School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China.
AMB Express. 2017 Dec;7(1):97. doi: 10.1186/s13568-017-0399-9. Epub 2017 May 15.
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11A, was selected from the mutagenesis library, E. coli/Aoxyn11A (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11A or AoXyn11A, was designed by replacing Tyr in AoXyn11A or AoXyn11A with Phe. Finally, three variant-encoding genes, Aoxyn11A , Aoxyn11A and Aoxyn11A , were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T ) of recombinant (re) AoXyn11A was 60 °C, being 5 °C higher than that of reAoXyn11A or reAoXyn11A, and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t ) of reAoXyn11A at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11A and reAoXyn11A. The melting temperature (T ) values of reAoXyn11A, reAoXyn11A, reAoXyn11A and reAoXyn11A were 52.3, 56.5, 58.6 and 61.3 °C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (k /K ) of reAoXyn11A was 473.1 mL mg s, which was 1.65-fold higher than that of reAoXyn11A.
为改善米曲霉糖苷水解酶家族（GHF）11木聚糖酶（AoXyn11A）的温度特性和催化效率，基于计算机辅助设计对其变体进行了预测。首先，通过对AoXyn11A三维结构的分子动力学（MD）模拟确定了具有最大B因子值的甘氨酸，对其进行了位点饱和诱变。因此，从诱变文库E. coli/Aoxyn11A（X：20种氨基酸中的任意一种）中筛选出了热稳定性最高的一个变体AoXyn11A。其次，基于AoXyn11A与7种嗜热GHF11木聚糖酶的一级结构多重比对，通过将AoXyn11A或AoXyn11A中的酪氨酸替换为苯丙氨酸来设计AoXyn11A或AoXyn11A。最后，采用两步全质粒PCR方法构建了3个变体编码基因Aoxyn11A、Aoxyn11A和Aoxyn11A，并分别在毕赤酵母GS115中表达。重组（re）AoXyn11A的最适温度（T）为60℃，比reAoXyn11A或reAoXyn11A高5℃，比reAoXyn11A高10℃。reAoXyn11A在50℃下的热失活半衰期（t）为240分钟，分别比reAoXyn11A、reAoXyn11A和reAoXyn11A长40倍、3.4倍和2.5倍。reAoXyn11A、reAoXyn11A、reAoXyn11A和reAoXyn11A的解链温度（T）值分别为52.3℃、56.5℃、58.6℃和61.3℃。这些发现表明，Gly21Ile和Tyr13Phe的迭代诱变以协同模式改善了AoXyn11A的温度特性。除此之外，reAoXyn11A的催化效率（k/K）为473.1 mL mg s，比reAoXyn11A高1.65倍。
