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化工学报  2015 

理性设计提高β-葡萄糖醛酸苷酶的热稳定性

DOI: 10.11949/j.issn.0438-1157.20141902, PP. 2205-2211

Keywords: β-葡萄糖醛酸苷酶,分子模拟,动力学,蛋白质稳定性,理性设计,同源比对,脯氨酸

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Abstract:

采用同源序列比对策略和脯氨酸效应的设计策略,以同源建模的三维结构为基础,结合定点突变技术,对重组产紫青霉β-葡萄糖醛酸苷酶(PGUS-E)进行理性设计,获得了2个热稳定性明显提高的突变体PGUS-EI130V和PGUS-EG280P,再将突变位点进行组合获得突变体PGUS-EI130V+G280P。相比PGUS-E,PGUS-EI130V、PGUS-EG280P和PGUS-EI130V+G280P在60℃下的半衰期T1/2分别比原始酶的23min提高3.5倍,5倍和5.5倍,达到82min,117min和128min。突变体的动力学参数Kcat/Km值分别为1.534×107mol-1·L·min-1,1.368×107mol-1·L·min-1和1.283×107mol-1·L·min-1,与原始酶(1.316×107mol-1·L·min-1)接近,对底物的亲和力基本不变。结果表明在蛋白质构象不稳定的区段中引入脯氨酸,以及在相应位置引入嗜热菌的氨基酸,均可提高蛋白质热稳定性。

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