Purification and characterization of a novel phloretin-2′-O-glycosyltransferase favoring phloridzin biosynthesis

Phloretin-2′- O -glycosyltransferase (P2′GT) catalyzes the last glycosylation step in the biosynthesis of phloridzin that contributes to the flavor, color and health benefits of apples and processed apple products. In this work, a novel P2′GT of Malus x domestica (Md P2′GT) with a specific activity of 46.82 μkat/Kg protein toward phloretin and uridine diphosphate glucose (UDPG) at an optimal temperature of 30 °C and pH 8.0 was purified from the engineered Pichia pastoris broth to homogeneity by anion exchange chromatography, His-Trap affinity chromatography and gel filtration. The purified Md P2′GT was low N -glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, Md P2′GT also exhibited activity towards quercetin and adenosine diphosphate glucose (ADPG), kaempferol and UDPG, quercetin and UDP-galactose, isoliquiritigenin and UDPG, and luteolin and UDPG, producing only one isoquercitrin, astragalin, hyperoside, isoliquiritin, or cynaroside, respectively. This broad spectrum of activities make Md P2′GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, Md P2′GT displayed the lowest K m and the highest k cat /K m for phloretin and UDPG compared to all previously reported P2′GTs, making Md P2′GT favor phloridzin synthesis the most.