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(RhaD)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RhaD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RhaD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RhaDY152A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RhaD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 (RhaD) 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 RhaD to exclusively produce D-psicose, site-saturation mutagenesis of tyrosine 152 of RhaD with other nineteen amino acids were performed. The results showed that D-psicose was favored by RhaD Y152A mutant and the ratio was increased eight folds. This study will provide theoretical foundation for further molecular modification of RhaD for the synthesis of D-psicose as a single product.