L-鼠李树胶糖-1-磷酸醛缩酶立体选择性的半理性改造合成D-阿洛酮糖

Semi-rational molecular engineering to improve stereoselectivity of L-rhamnulose-1-phosphate aldolase for D-psicose synthesis

稀有糖D-阿洛酮糖在食品、医药、保健等领域显示了巨大的应用潜能,然而以较高的收率大量生产D-阿洛酮糖依然面临着挑战。前期研究表明,属于磷酸二羟基丙酮(dihydroxyacetone phosphate,DHAP)依赖型醛缩酶家族的L-鼠李树胶糖-1-磷酸醛缩酶(Rha D)在以D-甘油醛为醛受体时,失去了对该醛受体的立体选择性,生成D-阿洛酮糖和D-山梨糖两种稀有糖,两者比例接近1∶1。为了对Rha D醛缩酶的立体选择性进行优化使其专一性地生产D-阿洛酮糖,对Rha D醛缩酶的152位酪氨酸进行了19种其他氨基酸的定点饱和突变,发现Rha DY152A突变体倾向于生成D-阿洛酮糖,D-阿洛酮糖和D-山梨糖的比例从最初的1∶1显著提高到8∶1,为下一步对Rha D醛缩酶分子改造进而定向合成单一产物D-阿洛酮糖提供理论依据。

Rare sugars D-psicose shows great potentials in many field,such as food,pharmaceutical and medicinal industries. However,large scale synthesis of D-psicose with a high yield still remains a challenge. In our previous study,DHAP-dependent aldolase L-rhamnulose-1-phosphate aldolase（ Rha D） from Escherichia coli lost its stereoselectivity when accepting D-glyceraldehyde as the acceptor. As a result,two rare sugars D-psicose and D-sorbose were generated with a ratio of 1∶ 1. In order to optimize the stereoselectivity of Rha D to exclusively produce D-psicose,sitesaturation mutagenesis of tyrosine 152 of Rha D with other nineteen amino acids were performed. The results showed that D-psicose was favored by Rha D Y152 A mutant and the ratio was increased eight folds. This study will provide theoretical foundation for further molecular modification of Rha D for the synthesis of D-psicose as a single product.