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Background: Several studies have reported that ginsenoside Rg3(<i>S</i>) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(<i>S</i>), which may improve nutritional activity, human health, and quality of life. Method: <i>Lactobacillus ginsenosidimutans</i> EMML 3041^T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(<i>S</i>) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(<i>S</i>), the presence of glycoside hydrolase in <i>Lactobacillus ginsenosidimutans</i> was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. Results: The <i>L. ginsenosidimutans</i> EMML 3041^T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (&#945;,&#946;-glucosidase and &#945;,&#946;-galactosidase) were cloned in <i>Escherichia coli</i> BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 &#176;C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1&#8594;Rd&#8594;Rg3(<i>S</i>). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(<i>S</i>) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(<i>S</i>) with 74.3% chromatography purity. Conclusion: Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(<i>S</i>) in the functional food and pharmaceutical industries.