A novel α-amylase from Streptococcus thermophilus 17140 with β-glycosidic bond hydrolysis capability for the transformation of rare ginsenosides
Alpha-amylases typically act on starch and oligosaccharides that contain α-1,4 glycosidic linkages, while ginsenosides primarily consist of β-1,2 and β-1,6 glycosidic bonds, which cannot be hydrolyzed by α-amylases. However, for the first time, we have successfully isolated Streptococcus thermophilus 17140 (St17140), capable of converting ginsenoside Rb1 into rare ginsenosides. St17140 expresses a novel α-amylase (StAMY), which hydrolyzes ginsenoside Rb1 to produce Rd, gypenoside XVII, F 2 , and Rh 2 . With Rb 1 as the substrate, the optimal reaction temperature is 50 °C and the optimal pH is 5.5. When Rb 1 , Rd, or gypenoside XVII are used as substrates, the Km are 0.135 mM, 0.0871 mM, and 0.260 mM respectively, the Kcat are 0.621 min −1 , 0.397 min −1 , and 0.297 min −1 respectively, and the Kcat/Km are 4.59 min −1 ·mM −1 , 4.56 min −1 ·mM −1 and 1.14 min −1 ·mM −1 respectively. Utilizing molecular docking and site-directed mutagenesis techniques, the mechanism of ginsenoside hydrolysis by StAMY was further elucidated. A107, F201, A337 and A268 are key amino acid residues that anchor the saponin in the active pocket, and D234 and D331 as catalytic amino acid residues to hydrolyze ginsenosides into rare ginsenosides. It is the first time to discover a novel α-amylase with the ability to hydrolyze ginsenosides β-1,2 and β-1,6 glycosidic linkage, and the first time to discover the ability of S. thermophilus to transform rare ginsenosides. These not only enrich the resources of glycosidases and offer novel insights for the rational modification of glycosidase, also are important for further elucidating the mechanism of probiotic transformation of rare ginsenosides and developing engineering bacteria with high production of rare ginsenosides.
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