Tier 4 — mechanistic

Fisetin glycosides synthesized by cyclodextrin glycosyltransferase from Paenibacillus sp. RB01: characterization, molecular docking, and antioxidant activity

Lorthongpanich N, Mahalapbutr P, Rungrotmongkol T, Charoenwongpaiboon T, Prousoontorn MH

PeerJ 2022 Volume 10, article e13467

Bibliography

PubMed: PMID 35637717
PubMed Central: PMC9147316
DOI: 10.7717/peerj.13467
Funding: 90th Anniversary of Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (grant no. GCUGR1125612074M).
Competing interests: The authors declare that they have no competing interests.

Study snapshot

Design	Enzymatic transglycosylation study using purified cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 with β-cyclodextrin as glycosyl donor, followed by product characterisation (TLC, HPLC, LC-MS/MS, enzymatic hydrolysis), molecular docking, and in vitro water-solubility and DPPH antioxidant assays.
Model	Cell-free enzymatic system: CGTase enzyme purified from Paenibacillus sp. RB01 bacterial culture; fisetin and β-cyclodextrin as substrates; homology-modelled CGTase active site for docking.
Sample	In vitro/analytical experiments performed in triplicate.
Intervention	Fisetin (0.25% w/v) and β-cyclodextrin (1.0% w/v) incubated with CGTase (200 U/mL) in DMSO-containing phosphate buffer (10-50% v/v DMSO) at 40°C for 24 hours to generate fisetin glycoside derivatives; purified fisetin monoglucoside products then tested for water solubility and DPPH radical-scavenging activity against parent fisetin and ascorbic acid.
Duration	24-hour enzymatic reaction; solubility and antioxidant assays performed after purification.
Endpoints	Glycoside product yield and structure (TLC, HPLC, LC-MS/MS fragmentation); Glycosidic linkage type (glucoamylase/α-glucosidase hydrolysis); Molecular docking binding energy and pose in the CGTase active site; Water solubility of purified fisetin monoglucosides vs parent fisetin; DPPH radical-scavenging activity (IC50) of fisetin monoglucosides vs parent fisetin and ascorbic acid

What the study showed, in plain terms

This is a chemistry and biotechnology paper, not a biological or clinical study — it is included in the Data Center because it is directly relevant to solving fisetin's biggest practical problem: it barely dissolves in water.

A Thai research team used a bacterial enzyme (CGTase, from a soil bacterium called Paenibacillus sp. RB01) to attach sugar molecules from cyclodextrin onto fisetin, creating several new 'fisetin glycoside' compounds. Think of it as chemically gluing a sugar tag onto fisetin to make it more water-friendly.

The two main fisetin-sugar compounds they purified (fisetin-7-O-glucoside and fisetin-4'-O-glucoside) dissolved in water at least 800 to 1,800 times better than plain fisetin, while keeping essentially the same antioxidant power (measured by DPPH radical scavenging) as unmodified fisetin.

This matters for the wider fisetin story because poor water solubility is repeatedly flagged across this Data Center as a key reason fisetin's promising lab findings do not always translate into strong effects when taken as an oral supplement. This paper demonstrates one laboratory-scale method (enzymatic glycosylation) that could, in principle, help address that gap — though it is a proof-of-concept chemistry study, not a tested supplement ingredient.

Key findings

CGTase from Paenibacillus sp. RB01 converted more than 400 mg/L of fisetin into glycoside derivatives, a substantially higher yield than a previously reported metabolically engineered E. coli method.
At least five distinct fisetin glycoside products were identified by HPLC and LC-MS/MS, including fisetin mono-, di- and tri-glucosides and positional isomers, linked via α-1,4-glycosidic bonds.
The two purified fisetin monoglucosides were structurally identified as fisetin-7-O-glucoside and fisetin-4'-O-glucoside by MS/MS fragmentation pattern analysis, consistent with molecular docking predictions of two possible glycosylation sites on the fisetin molecule.
Water solubility of fisetin-7-O-glucoside and fisetin-4'-O-glucoside was increased by at least 1,800-fold and 888-fold respectively compared with parent fisetin (0.46 µM soluble).
DPPH antioxidant activity was comparable between the fisetin glycosides (IC50 2.28 and 2.52 µM) and parent fisetin (IC50 2.72 µM), indicating the glycosylation did not meaningfully compromise antioxidant capacity, unlike glycosylation of some other flavonoids reported in the literature.

What this study can and cannot tell us

This is a cell-free enzymatic chemistry study; no cell, animal or human data are presented, so nothing can be concluded here about bioavailability, absorption or biological efficacy of fisetin glycosides in a living organism.

Fisetin di- and tri-glucoside yields were relatively low, so solubility and antioxidant testing were only performed on the two monoglucoside products; the properties of the higher-order glycosides remain uncharacterised.

CGTase enzyme activity dropped sharply above 40% (v/v) DMSO co-solvent and was almost fully lost at 50%, constraining the reaction conditions and overall conversion efficiency (17% of supplemented fisetin converted under optimal conditions).

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