title: Purification and characterization of a novel phloretin-2'-O-glycosyltransferase favoring phloridzin biosynthesis.
abstract: 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 (MdP2'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 MdP2'GT was low N-glycosylated and secreted as a stable dimer with a molecular mass of 70.7 kDa in its native form. Importantly, MdP2'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 MdP2'GT a promising biocatalyst for the industrial preparation of the corresponding polyphenol glycosides, preferably for their subsequent isolation and purification. Besides, MdP2'GT displayed the lowest Kmand the highest kcat/Kmfor phloretin and UDPG compared to all previously reported P2'GTs, making MdP2'GT favor phloridzin synthesis the most.
authors.affiliation: College of Food Science and Engineering, Northwest A&F University, Yang ling, Shaanxi, 712100, China.
authors.forename: Tingjing
authors.initials: T
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Liang
authors.affiliation: Department of Food Science, University of Massachusetts, Amherst, MA01003, USA.
authors.forename: Panxue
authors.initials: P
authors.lastname: Wang
authors.affiliation: College of Life Science and Engineering, Shaanxi University of Science &Technology, Xi'an, Shaanxi, 710021, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.affiliation: College of Food Science and Engineering, Northwest A&F University, Yang ling, Shaanxi, 712100, China.
authors.forename: Yutang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Food Science and Engineering, Northwest A&F University, Yang ling, Shaanxi, 712100, China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Wei
authors.affiliation: College of Food Science and Engineering, Northwest A&F University, Yang ling, Shaanxi, 712100, China.
authors.forename: Mingtao
authors.initials: M
authors.lastname: Fan
mesh_terms: D006031:Glycosylation; D016695:Glycosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010693:Phloretin / Q000378:metabolism*; D010695:Phlorhizin / Q000096:biosynthesis*
chemical_list: D010695:Phlorhizin; D016695:Glycosyltransferases; D010693:Phloretin