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

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by α-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTase-glysosyl intermediate, in which O7 and O4’ atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids

Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control

Fructooligosaccharides (FOSs) are well-known prebiotics that are widely used in the food, beverage and pharmaceutical industries. Inulosucrase (E.C. 2.4.1.9) can potentially be used to synthesise FOSs from sucrose. In this study, inulosucrase from Lactobacillus reuteri 121 was engineered by site-directed mutagenesis to change the FOS chain length. Three variants (R483F, R483Y and R483W) were designed, and their binding free energies with 1,1,1-kestopentaose (GF4) were calculated with the Rosetta software. R483F and R483Y were predicted to bind with GF4 better than the wild type, suggesting that these engineered enzymes should be able to effectively extend GF4 by one residue and produce a greater quantity of GF5 than the wild type. MALDI-TOF MS analysis showed that R483F, R483Y and R483W variants could synthesise shorter chain FOSs with a degree of polymerization (DP) up to 11, 10, and 10, respectively, while wild type produced longer FOSs and in polymeric form. Although the decrease in catalytic activity and the increase of hydrolysis/transglycosylation activity ratio was observed, the variants could effectively synthesise FOSs with the yield up to 73% of substrate. Quantitative analysis demonstrated that these variants produced a larger quantity of GF5 than wild type, which was in good agreement with the predicted binding free energy results. Our findings demonstrate the success of using aromatic amino acid residues, at position D418, to block the oligosaccharide binding track of inulosucrase in controlling product chain length