定向进化提高嗜热拟青霉J18耐热β-1,3-1,4-葡聚糖酶在酸性条件下的催化能力

Improvement of catalytic capability of Paecilomyces thermophila J18 thermostable β-1,3-1,4-glucanase under acidic condition by directed evolution

应用定向进化技术提高了嗜热拟青霉Paecilomyces thermophila J18耐热β-1,3-1,4-葡聚糖酶(PtLic16A)在酸性条件下的催化能力。结合易错PCR和DNA改组的方法,构建了β-葡聚糖酶的突变体文库;利用刚果红染色法建立了阳性克隆的高通量筛选体系。筛选得到的突变酶PtLic16AM1的反应最适pH由7.0变化至5.5,且保持了原有的耐热性和比酶活。突变酶的DNA序列中有4个点位发生突变,引发了4处氨基酸替换,分别是T58S、Y110N、G195E和D221G。结构模拟结果显示,发生突变的4个氨基酸位点中,Y110N位置靠近酶活性中心,而T58S、G195E和D221G则离酶活性中心较远,其中T58S、G195E可能对酶最适pH的变化起到了关键作用。

Directed evolution was used to improve the performance of β-l,3-1,4-glucanase （designated as PtEicl6A） from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant （PtLicl6AM1） exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLicl6AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sensesubstitutions which caused four amino acid substitutions - namely T58S, Y11 ON, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y11 ON was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable β-1,3-1,4-glucanase for industrial use.