基于非理性设计的肺炎克雷伯氏杆菌甘油脱水酶的分子定向进化

Irrational Design Directed Evolution of Glycerol Dehydratase of Klebsiella pneumoniae in vitro

1,3-丙二醇是一种具有广泛应用前景和巨大市场潜力的平台化合物。在生物体转化甘油合成1,3-丙二醇的代谢途径中,甘油脱水酶是代谢途径的限速酶,然而自然界中的甘油脱水酶却存在着诸多的不足。本研究采用易错PCR技术对来源于肺炎克雷伯氏杆菌的甘油脱水酶进行非理性设计的分子定向进化,通过高通量筛选方法筛选获得了一个酶学性质得到了改善的突变酶γ-F97G。当以甘油作为酶催化的底物时,突变酶的酶比活力为(583.8±49.4)U/mg,相比较野生型的酶比活力提高了约94%;当以1,2-丙二醇作为酶催化底物时,突变酶的酶比活力为(147.9±5.5)U/mg,相比较野生型的酶比活力提高了约72%。经分析酶蛋白的三维结构后发现酶蛋白发生突变的位点属于远距离突变位点,实验结果表明远距离突变有时同样是一条优化和改善酶的酶学性质的好途径。

1,3-Propanediol, as a platform compounds, has been widely used in many industries, which shows the huge market potential. Glycerol dehydratase plays an important role in the biological pathways for converting glycerol to 1,3-propanediol, it is the rate-limiting enzyme in this synthesis process, but it has some disadvantages. In this study, Error-Prone PCR was applied to evolve glycerol dehydratase and screened the library of glycerol dehydratase mutants by the high throughput screening method. We successfully screened one property optimized mutant γ-F97G. For glycerol as substrate, the specific activity of γ-F97G was （583.8±49.4） U/rag, which was enhanced about 94% than that of wild type. Meanwhile, for 1,2-propanediol as substrate, the specific activity of γ-F97G was （147.9±5.5） U/mg, which was enhanced about 72% than that of wild type. On basis of the analysis of three-dimensional structure model of mutant γ-F97G, this study found that the mutated site of mutant γ-F97G was distant from the active center, which supplied the further evidence that distant mutations could be a good approach for evolving enzyme, too.