March 9, 2021 Longchang Chemical

Saponins are a class of glycosides in which aglycones are triterpene or sterane compounds. They are one of the effective ingredients of many Chinese herbal medicines such as ginseng, licorice and yam (the main saponins structure is shown in Figure 1). Enhance immunity and other functions. There are many reports on the biotransformation of ginsenosides in the literature. At present, more than 150 kinds of ginsenosides have been separated and identified. The contents of ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1 are as high as 80%, while the contents of ginsenosides Rg3, Rh2, F2 and Compound K (C-K) and other rare saponins have little or no content. Studies have shown that some rare saponins have good pharmacological activities. However, due to the low content, preparation and production are restricted. The same type of ginsenoside has the same aglycon, but the sugar chain is different. Rare ginsenosides and the higher content of the same type of saponins often only differ by 2 to 3 sugar groups. Therefore, the same type of active rare saponin can be prepared by enzymatic hydrolysis of high-content saponin.

Figure 1. Structure of main saponins

Different glycoside hydrolases have different selectivities, and the pathways to hydrolyze ginsenosides are also different. As shown in Table 1, different glycoside hydrolases can be used to prepare different rare ginsenosides. Ginsenoside Rd can be prepared by hydrolyzing ginsenosides Rb1, Rb2, Rb3 and Rc’s C-20 outer sugar group. The β-glucosidase isolated and purified from China white jade snail and Thermus caldophilus can convert ginsenoside Rb1 to Rd. Kim et al. obtained it from soil microorganisms using molecular cloning technology to convert ginsenoside Rb1 which is the recombinant glycoside hydrolase of Rd. Subsequently, researchers cloned glucosidase from Thermotoga thermarum and Bifidobacterium longum H-1, which improved the efficiency of transformation and preparation of ginsenoside Rd. The glucosidase obtained from Flavobacterium johnsoniae and Thermus thermophilus by recombinant technology can not only convert ginsenoside Rb1 into Rd, but also hydrolyze the C-20 sugar chain of Gypenoside XVII (G17) to produce ginsenoside F2. In addition to glucosidase, α-L-arabinofuranoside hydrolase that can convert ginsenoside Rc to Rd was obtained from ginseng root and Leuconostoc sp. Α-L-arabinofuranoside hydrolase and α-L-arabinopyranoside hydrolase are obtained from Bifidobacterium breve and Bifidobacterium longum, which can transform ginsenoside Rc and Rb2 into Rd. It has been reported in the literature that the α-L-arabinofuranoside hydrolase in Caldicellulosiruptor saccharolyticus and Rhodanobacter ginsenosidimutans can not only hydrolyze ginsenoside Rc to Rd, but also convert Compound Mc1 (C-Mc1) into F2. The glycoside hydrolase isolated and purified from Aspergillus by Yu et al. can convert all ginsenosides Rb1, Rb2, Rb3 and Rc into Rd. Some glycoside hydrolases can completely hydrolyze the sugar chains at the C-20 position in molecules such as glycol-type ginsenosides Rb1, Rb2, Rb3, Rc and Rd, to generate ginsenoside Rg3, which enables large-scale production of Rg3 and is developed as an anti-tumor agent drug. The glucosidase in Paecilomyces bainier and Microbacterium esteraromaticum can directly hydrolyze ginsenoside Rb1 into Rg3, while the glucosidase isolated and purified from Microbacterium esteraromaticum can hydrolyze ginsenoside Rb2 into Rg3. The recombinant glycoside hydrolase cloned from Pseudonocardia by molecular cloning technology can transform ginsenosides Rb1, Rb3 and Rd to prepare Rg3. Similarly, a series of active rare ginsenosides can be prepared by hydrolyzing the sugar group at position C-3 in ginsenosides. The recombinant glucosidase cloned from Sphingomonas and Sphingopyxis alaskensis can hydrolyze the glucose outside the sugar chain at position C-3 in the ginsenoside Rb1, Rb2, Rc, Rd, and Rg3 molecules, and prepare G17, Compound O (CO), and C-Mc1, F2 and Rh2. Some glycosidases can directly hydrolyze the inner glucosyl group at the C-3 position. For example, the glucosidase from Terrabacter ginsenosidimutans and Esteya vermicola can hydrolyze the sugar chain at the C-3 position of the ginsenoside Rb1, Rb2, Rb3, Rc and Rd molecules to produce The corresponding saponin LXXV (G75), Compound Y (C-Y), Compound Mx (C-Mx), Compound Mc (C-Mc) and C-K. In addition, some glycoside hydrolases can simultaneously hydrolyze the C-20 and C-3 sugar groups in glycol-type ginsenosides. The recombinant glucosidase cloned from Arthrobacter chlorophenolicus can convert ginsenosides Rb1, Rb2 and Rc into F2. The glycoside hydrolase in Fusobacterium K60, endophytic fungi GE 17-18, Sulfolobus acidocaldarius, Aspergillus niger and Microbacteriu esteraromaticum can hydrolyze ginsenoside Rb1 to produce C-K.

Table 1. Biotransformation of ginsenosides by glycosidase

ProductSubstrateReactionOrganism
RdRb1β-GlucosidaseChina white jade snail
RdRb1β-GlucosidaseThermus caldophilus
RdRb1β-GlucosidaseUncultured bacteria
RdRb1β-GlucosidaseThermotoga thermarum
RdRb1β-GlucosidaseBifidobacterium longum H-1
RdRb1β-GlucosidaseFlavobacterium johnsoniae
RdRb1β-GlucosidaseThermus thermophilus
RdRb1β-GlucosidasePenicillium oxalicum
RdRb1β-GlucosidaseCladosporium fulvum
RdRcα-L-ArabinofuranosidasePanax ginseng
RdRcα-L-ArabinofuranosidaseLeuconostoc
RdRcα-L-ArabinofuranosidaseBifidobacterium breve
RdRcα-L-ArabinofuranosidaseBifidobacterium longum
RdRcα-L-ArabinofuranosidaseCaldicellulosiruptor saccharolyticus
RdRcα-L-ArabinofuranosidaseRhodanobacter ginsenosidimutans
RdRb2α-L-ArabinopyranosidaseBifidobacterium breve
RdRb2α-L-ArabinopyranosidaseBifidobacterium longum
RdRb1/Rb2/Rb3/RcGlycosidaseAspergillus
Rg3Rb1β-GlucosidasePaecilomyces bainier
Rg3Rb1β-GlucosidaseMicrobacterium esteraromaticum
Rg3Rb2β-GlucosidaseMicrobacterium esteraromaticum
Rg3Rb1/Rb3/Rdβ-GlucosidasePseudonocardia
G17Rb1β-GlucosidaseSphingomonas
G17Rb1β-GlucosidaseSphingopyxis alaskensis
G17Rb1β-GlucosidaseCellulosimicrobium cellulans
G75Rb1β-GlucosidaseTerrabacter ginsenosidimutans
G75Rb1β-GlucosidaseEsteya vermicola
F2G17β-GlucosidaseFlavobacterium johnsoniae
F2G17β-GlucosidaseThermus thermophilus
F2C-Mc1α-L-ArabinofuranosidaseCaldicellulosiruptor saccharolyticus
F2C-Mc1α-L-ArabinofuranosidaseRhodanobacter ginsenosidimutans
F2Rdβ-GlucosidaseCellulosimicrobium cellulans
F2Rb1/Rb2/Rcβ-GlucosidaseArthrobacter chlorophenolicus
Rh2Rg3β-GlucosidaseSphingopyxis alaskensis
CKRdβ-GlucosidaseTerrabacter ginsenosidimutans
CKRdβ-GlucosidaseEsteya vermicola
CKRb1β-GlucosidaseFusobacterium K-60
CKRb1β-Glucosidaseendophytic fungi GE 17-18
CKRb1/Rb2β-GlucosidaseSulfolobus acidocaldarius
CKRb1/Rb2/Rb3/Rcβ-GlucosidaseAspergillus niger
CKRb1/Rb2β-GlucosidaseMicrobacteriu esteraromaticum
C-ORb2β-GlucosidaseCellulosimicrobium cellulans
C-YRb2β-GlucosidaseTerrabacter ginsenosidimutans
C-McRcβ-GlucosidaseTerrabacter ginsenosidimutans
C-Mc1Rcβ-GlucosidaseCellulosimicrobium cellulans
C-MxRb3β-GlucosidaseTerrabacter ginsenosidimutans
Rg2Reβ-GlucosidaseMicrobacterium esteraromaticum
Rg2Reβ-GlucosidaseMucilaginibacter
Rg2Reβ-GlucosidasePseudonocardia
Rh1Rg1β-GlucosidaseMicrobacterium esteraromaticum
Rh1Rfβ-GlucosidasePyrococcus furiosus
Rh1Rfβ-GlucosidaseAspergillus niger
Rh1Rg2α-L-RhamnosidaseAbsidia
Rh1R2β-XylosidaseThermoanaerobacterium
F1Rg1β-GlucosidaseFusarium moniliforme
F1Rg1β-GlucosidasePenicillium sclerotiorum
F1Rg1β-GlucosidaseSanguibacter keddieii

G17: gypenoside XVII; G75: gypenoside LXXV; C-O: compound O; C-Y: compound Y; C-Mc1: compound Mc1; C-Mc: compound Mc; C-Mx: compound Mx; C-K: compound K.

The C-6 and C-20 sugar groups in triol ginsenosides can also be hydrolyzed by glycoside hydrolases. Ginsenoside Rg2 can be obtained by hydrolyzing C-20 glucose in Re molecule by glycosidase. The recombinant glucosidase cloned from Microbacterium esteraromaticum, Mucillaginibacter and Pseudonocardia can not only convert ginsenoside Re into Rg2, but also Ginsenoside Rg1 is converted into Rh1. Glucose, rhamnose and xylose outside the C-6 position of ginsenoside Rf, Rg2 and R2 can all be converted to prepare Rh1. Unlike ginsenoside Rh1, ginsenoside F1 has only one glucose attached to the C-20 position of its aglycon. Glucosidase in Fusarium moniliforme, Penicillium sclerotiorum and Sanguibacter keddieii can specifically hydrolyze the C-6 glucose of ginsenoside Rg1 to produce ginsenoside F1.

Glycoside hydrolase is not only used to transform and prepare active rare ginsenosides, but also is widely used to hydrolyze and modify saponins such as licorice, soybean and yam (Table 2). Glucuronidase isolated and purified from Streptococcus LJ-22 and Penicillium purpurogenum Li-3 can hydrolyze glycyrrhizin to produce monoglucuronic acid glycyrrhizin, and there is no by-product glycyrrhetinic acid. Morana et al. used glucuronidase derived from Aspergillus niger to completely hydrolyze glycyrrhizin to produce glycyrrhetinic acid. Soyasaponin hydrolase isolated and purified from Aspergillus oryzae can hydrolyze soyasaponin I to produce soyasaponol B. A new soy saponin hydrolase in Neocosmospora vasinfecta can convert soy saponin I, II, and III into soy saponin B, which provides an effective tool for preparing soybean saponin with anti-oxidation and blood lipid regulation. Among the steroidal saponins, the research and comparison of the hydrolysis modification of the sugar chain of dioscin is systematic. Inoue et al. isolated and purified a glucosidase from Costus speciosus that can hydrolyze the original diosgenin to produce diosgenin. Liu et al. isolated, purified and cloned from Aspergillus oryzae to obtain a recombinant dioscin hydrolase, which can hydrolyze the glucosyl and α-1,4 rhamnosyl groups in dioscin to produce dioscin III. The α-L-rhamnosidase isolated and purified by Feng et al. from Curvularia lunata can hydrolyze the α-1,2 rhamnosyl group in dioscin to produce dioscin V. Qian et al. isolated and purified an α-L-rhamnosidase from fresh beef liver, which can hydrolyze the α-1,2 and α-1,4 two rhamnosyl groups in diosgenin to form a glucose group. -Diosgenin. Fu et al. isolated and purified diosgenin hydrolase from Absidia, which can completely hydrolyze diosgenin into diosgenin.

Table 2. Biotransformation of other saponins by glycosidase

ProductSubstrateReactionOrganism
GAMGGlycyrrhizinβ-GlucuronidaseStreptococcus
GAMGGlycyrrhizinβ-GlucuronidasePenicillium purpurogenum
Glycyrrhetinic acidGlycyrrhizinβ-GlucuronidaseAspergillus niger
Soyasapogenol BSoyasaponin ISoybean saponin hydrolaseAspergillus oryzae
Soyasapogenol BSoyasaponinSoybean saponin hydrolaseNeocosmospora vasinfecta
DioscinProtodioscinβ-GlucosidaseCostus speciosus
Progenin IIIProtodioscinProtodioscin-glycosidaseAspergillus oryzae
Progenin VDioscinα-L-RhamnosidaseCurvularia lunata
Diosgenyl-glucosideDioscinα-L-RhamnosidaseBovine liver
DiosgeninDioscinDioscin-glycosidaseAbsidia

GAMG: Glycyrrhetic acid mono-glucuronide

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