E49F对麦氏交替单胞菌木聚糖酶XynZT-2的活性分析

Effect of E49F on the activity of xylanase XynZT-2 from Alteromonas macleodii

为了探究酶表面氨基酸残基对催化性能的影响,对麦氏交替单胞菌(Alteromonas macleodii)的木聚糖酶XynZT-2进行分子改造。基于计算机模拟,预测了潜在的有益突变体E49F,通过定点突变构建突变酶基因xynZT-2E49F,并将原酶与突变酶基因转化大肠杆菌BL21(DE3)异源表达。酶学性质分析发现,突变酶XynEF最适温度为70℃,相比原酶XynZT-2提高了25℃,酶活力提高了6.4倍。酶动力学分析发现,突变酶的k cat/K_(m)值相比原酶提高了10.1倍,突变酶的K_(m)值明显下降,对底物的亲和力增强。通过酶与底物分子对接,揭示了底物在突变酶催化活性口袋内的结合构象以及酶活力提高的潜在原因。为GH43家族木聚糖酶的分子改造研究提供了基础。

To explore the effect of amino acid residues above enzyme surface on the catalytic properties,the molecular modification of xylanase XynZT-2 from Alteromonas macleodii was designed.Based on the computer simulation,the potential beneficial mutant E49F was predicted,and the mutant enzyme gene xynZT-2 E 49 F was constructed by site-directed mutagenesis.Then the proenzyme and mutant enzyme gene were transformed into E.coli BL21(DE3)for heterologous expression.Enzymic properties analysis showed that the optimum temperature of the mutant enzyme XynEF was 70℃,which was 25℃higher than that of the original XynZT-2,and the enzyme activity of XynEF was 6.4 times higher than that of XynZT-2.The k cat/K_(m) value of the mutant enzyme was 10.1 times higher than that of the original enzyme.The K_(m) value of the mutant enzyme was significantly decreased,and the affinity of the mutant enzyme to the substrate was enhanced.The binding conformation of the substrate in the catalytic activity pocket of the mutant enzyme and the potential reason for the improvement of enzyme activity were revealed by the molecular docking of the enzyme with the substrate molecule.It provides a basis for the molecular modification of GH43 family xylanase.