Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillus vitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis

Abstract

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0–9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10−3 mM s−1), and Kcat/Km (763.92 mM−1 s−1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.

Mussel-Inspired Electro-oxidation-Modified Three-Dimensional Printed Carriers for a Versatile Enzyme Immobilization Approach

“…The synthesis of isofloridoside catalyzed by α-galactosidase galV was performed according to previous research with appropriate modifications . Galactose (0.3 M), glycerol (3 M), and 50 U of free galV or 3DABS-PDA@galV were mixed, and 50 mM Na 2 HPO 4 –NaH 2 PO 4 (pH = 7.5) buffer was added to bring the final volume of the reaction solution to 10 mL.…”

A GH42 β‐galactosidase from Bifidobacterium thermophilum: Biochemical characterization and synthesis of galactosyl glycerol by reverse hydrolysis

“…Galactosyl glycerol is a natural glycoside composed of d ‐galactopyranosyl and glycerol, which is found in various plants and algae, especially in photosynthetic tissues 21 . Galactosyl glycerol has diverse applications in food, healthcare products, cosmetics, and even medicine 19,21–23 . Additionally, glycoglycerolipids have remarkably attracted scholars’ attention due to their various biological functions, such as modulating fertility, as well as antibacterial, anti‐inflammatory, and antitumor activities.…”

“…Thus, galactosyl glycerol is an important chemical for the synthesis of new functional glycerolipids 24 . To date, few studies have concentrated on the enzymatic synthesis of galactosyl glycerol by the reverse hydrolysis 19,21,23 . Therefore, screening and discovering a new β‐galactosidase with a high condensation capability is of paramount importance for the establishment of an efficient reverse hydrolysis system for galactosyl glycerol preparation.…”

“…21 Galactosyl glycerol has diverse applications in food, healthcare products, cosmetics, and even medicine. 19,[21][22][23] Additionally, glycoglycerolipids have remarkably attracted scholars' attention due to their various biological functions, such as modulating fertility, as well as antibacterial, anti-inflammatory, and antitumor activities. Glycosylation is a key step in the synthesis of glycoglycerolipids.…”

“…24 To date, few studies have concentrated on the enzymatic synthesis of galactosyl glycerol by the reverse hydrolysis. 19,21,23 Therefore, screening and discovering a new β-galactosidase with a high condensation capability is of paramount importance for the establishment of an efficient reverse hydrolysis system for galactosyl glycerol preparation. In the present study, a GH42 β-galactosidase encoding gene btgal42 was cloned from Bifidobacterium thermophilum NJ-5 that was isolated from dairy farm soil.…”