Identification and Characterization of the Rhizobium sp. Strain GIN611 Glycoside Oxidoreductase Resulting in the Deglycosylation of Ginsenosides

Abstract

Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous.

Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily

“…This unusual catalytic mechanism related to the oxidation of sugar moiety gave an inspiration to the identification and characterization of our FAD-dependent glycoside oxidoreductase (FAD-GO) reaction from R sp. GIN611 20 , which was reported from our previous research. The FAD-GO did not show the dependency of NAD + and divalent metal ion unlike the enzymes in family GH4.…”

“…sp. GIN611 20 . The pETDuet-1-S vector was used for the construction of the plasmids varying oxidoreductase genes ( Agrobacterium tumefaciens str.…”

Identification and functional expression of genes encoding flavonoid O‐ and C‐glycosidases in intestinal bacteria

“…The enzyme system in strain CG19-1 also shares some features with a flavin adenine dinucleotide-dependent glycoside oxidoreductase that cleaves ginsenoside O-glucosides from Rhizobium sp. strain GIN611 (Kim et al, 2012). Two proteins were reported to participate in ginsenoside deglycosylation, the smaller of which enabled the expression of the oxidoreductase in a soluble form.…”

Comparative Genomics and CAZyme Genome Repertoires of Marine Zobellia amurskyensis KMM 3526T and Zobellia laminariae KMM 3676T

“…The GMC oxidoreductases are a very large and functionally diverse enzyme superfamily divided into four subfamilies, which include cellobiose dehydrogenases, glucose oxidoreductases, aryl-alcohol oxidases, alcohol oxidases, and pyranose oxidoreductases [63]. In 2012, Kim et al [64] identified GMC oxidoreductase from a Rhizobium sp. strain GIN611 with glycoside deglycosylation activity different from that of common glycosidases (GHs).…”