Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillus
vitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis |
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Authors: | Jialing Wang Xuefei Cao Weihao Chen Jiaxing Xu Bin Wu |
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Affiliation: | 1.College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, 30 Puzhunan Road, Nanjing 211816, China; (J.W.); (X.C.); (W.C.);2.School of Pharmaceutical Sciences, Nanjing Tech University, 30 Puzhunan Road, Nanjing 211816, China;3.College of Chemistry and Chemical Engineering, Huaiyin Normal University, 111 Jiangxi Road, Huai’an 223300, China |
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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. |
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Keywords: | α -Galactosidase Anoxybacillus vitaminiphilus thermal-stability reverse hydrolysis isofloridoside |
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