Characterization of the (Engineered) Branching Sucrase GtfZ-CD2 from Apilactobacillus kunkeei for Efficient Glucosylation of Benzenediol Compounds

Branching sucrases, a subfamily of Glycoside Hydrolase family (GI-170), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (alpha 1 -> 3) branching sucrase GtfZ-CD2 from Apilactobacillus kunkeei DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e., catechol, resorcinol, and hydroquinone) to form monoglucoside and diglucoside products. The production and yield of catechol glucosylated products were significantly higher than that of resorcinol and hydroquinone, revealing a preference for adjacent aromatic hydroxyl groups in glucosylation. Amino residues around acceptor substrate binding subsite +1 were targeted for semirational mutagenesis, yielding GtfZ-CD2 variants with improved resorcinol and hydroquinone glucosylation. Mutant L1560Y with improved hydroquinone mono-glucosylated product synthesis allowed enzymatic conversion of hydroquinone into alpha-arbutin. This study thus revealed the high potential of GH70 branching sucrases for glucosylating noncarbohydrate molecules. IMPORTANCE Glycosylation represents one of the most important ways to expand the diversity of natural products and improve their physico-chemical properties. Aromatic polyphenol compounds widely found in plants are reported to exhibit various remarkable biological activities; however, they generally suffer from low solubility and stability, which can be improved by glycosylation. Our present study on the glucosylation of benzenediol compounds by GH70 branching sucrase GtfZ-CD2 and its semirational engineering to improve the glucosylation efficiency provides insight into the mechanism of acceptor substrates binding and its glucosylation selectivity. The results demonstrate the potential of using branching sucrase as an effective enzymatic glucosylation tool.

Automated summary

This study revealed the potential of GH70 branching sucrases for glucosylating noncarbohydrate molecules. The (alpha 1 -> 3) branching sucrase GtfZ-CD2 was found to glucosylate benzenediol compounds and showed a preference for adjacent aromatic hydroxyl groups. Semirational engineering of GtfZ-CD2 variants improved the glucosylation efficiency of resorcinol and hydroquinone. This research provides insight into the mechanism of acceptor substrates binding and demonstrates the potential of using branching sucrase as an effective enzymatic glucosylation tool.