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Classification of Ginsenosidases
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This entry is adapted from the peer-reviewed paper 10.3390/foods12020397

Although there are multiple catalytic strategies for the conversion of major ginsenoside, biotransformation using ginsenosidases is more advantageous for the targeted production of minor ginsenosides due to the high specificity, high selectivity, high catalytic efficiency, high product purity, mild reaction conditions and clear catalytic process, which is one of the current research topics in ginsenoside biotransformation. Ginsenosidases are commonly classified as type I, type II, type III, type IV and type V according to the hydrolysis site, residues and types of sugar moieties of ginsenosides. Ginsenosidase type I simultaneously hydrolyze the glycosyl residues linked to C-20 and C-3 positions in the PPD-type ginsenosides, yielding minor ginsenosides with only one glucose residue or other glycosyl residues. Ginsenosidase type II hydrolyzes the glycosyl residues at the C-20 position of protopanaxadiol (PPD)-and protopanaxatriol (PPT)-type saponins. Ginsenosidase type III hydrolyzes the sugar moieties attached to the C-3 position of PPD-type ginsenosides. Ginsenosidase type IV only hydrolyzes the sugar moieties linked to C-6 in PPT-type ginsenosides. Ginsenosidase type V hydrolyzes the glycosyl moieties attached to the C-20 and C-6 positions of PPT-type ginsenosides to yield the corresponding aglycones.

ginsenoside Rb1 β-glucosidase minor ginsenoside bioconversion

1. Introduction

The Asian species Panax ginseng and Panax notoginseng, as well as the American ginseng Panax quinquefolius, are widely produced in China, Korea and North America due to their great bioactive value. Their dried roots and rhizomes have a long history of use as functional foods, medicine and dietary supplements, and the global ginseng market is expected to reach USD 900 million in 2027 [1].
The active ingredients in ginseng plants include ginsenosides, polysaccharides, amino acids, volatile oil, polyacetylenes, sterols, flavonoids, etc. [2]. Ginsenosides, of which more than 200 kinds have been identified to date, are the main bioactive ingredients of ginseng [3]. The outstanding pharmacological effects of ginsenosides include anti-tumor, anti-diabetic, anti-inflammatory, anti-allergic, immunomodulatory, hepatoprotective effect, neuroprotective effect, etc. (Figure 1) [4][5][6][7][8]. Chemically, ginsenosides are triterpene saponins, and they can be classified as dammaranes and oleananes-type triterpenes based on their aglycone skeletons. Dammarane-type ginsenosides are the main active ingredients and they are further classified into protopanaxadiol (PPD) type with hydroxyls at the 3β and 12β in the nucleus and protopanaxatriol (PPT) type with hydroxyls at the 3β, 12β and 6α in the nucleus [9]. The ginsenosides Rb1, Rb2, Rc, Re, Rd and Rg1, whose side chains have carbohydrate moiety with several monosaccharide residues, comprise more than 80% of the total ginsenosides in wild ginseng, and are considered to be the major ingredients [10][11]. By contrast, the minor ginsenosides F2, CK, Rg3, Rh2 and aglycon PPD (APPD). constitute less than 1% of total ginsenosides in wild ginseng [3].
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Figure 1. The biological activities of deglycosylated ginsenosides from Rb1. The picture materials were downloaded from Vecteezy (https://www.vecteezy.com, accessed on 11 November 2022) and Smart (https://smart.servier.com, accessed on 11 November 2022), which provide free pictures.
The content of ginsenoside Rb1 among total ginsenosides reaches approximately 20% [12]. However, ginsenoside Rb1 has poor membrane permeability, and it is comparatively easily excreted by the biliary tract and urinary system because of its dammarane tetracyclic triterpenoid skeleton, as well as the high number of flexible side-chain glycosyl moieties [13][14][15]. Pharmacokinetic studies in rats have shown that the oral bioavailability of ginsenoside Rb1 is only 0.1–4.35% [16]. The biotransformation by intestinal microbiota results in a decrease in the number of side-chain glucose residues, so after entering the organism, the major ginsenosides Rb1, Rc, etc., are transformed into the deglycosylated minor ginsenosides such as CK and Rh2 [17][18]. Compared to glycosylated ginsenosides, minor ginsenosides are more easily absorbed from the gastrointestinal tract into the bloodstream, whereby CK, Rh2 and APPD are much more toxic to tumor cells than the major ginsenosides, as shown in Figure 1, suggesting that minor ginsenosides are the main bioactive saponins of ginseng [19].
At present, there are three main biocatalytic strategies for the conversion of ginsenoside Rb1. One is the direct use of bacterial or fungal strains/whole cells to ferment ginsenoside Rb1. The second strategy is to first separate the crude β-glucosidase secreted during the growth of bacteria or fungi by methods such as ammonium sulfate precipitation, and further obtain the purified β-glucosidase by protein separation methods such as dialysis and column chromatography, and finally hydrolyze ginsenoside Rb1 using these purified β-glucosidases. The third strategy is to obtain pure β-glucosidase to enzymatically hydrolysis ginsenoside Rb1 by heterologous expression and purification by affinity chromatography and other methods. The production of minor ginsenosides using microbial strains/whole cells is generally an efficient and inexpensive process, but it is accompanied by the disadvantages of low specificity, long conversion time, unclear enzymes and poor access to intermediate prosapogenins. By contrast, biotransformation using purified enzymes and recombinant enzymes is more advantageous for the targeted production of minor ginsenosides due to the high specificity, high selectivity, high catalytic efficiency, high product purity, mild reaction conditions and clear catalytic process, which is one of the current research topics in ginsenoside biotransformation [20].
β-Glucosidase (EC 3.2.1.21) releases non-reducing terminal glucosidic residues from glycosylated metabolites or oligosaccharides [21], and it acts mainly on β-1,4 glucosidic bonds, in addition to β-1,2 bonds, β-1,3 bonds and β-1,6 bonds. The side-chain glycosyl moieties of ginsenoside Rb1 are connected through β-1,2 bonds and β-1,6 bonds, which can be effectively hydrolyzed by some β-glucosidases to bioconvert major ginsenoside Rb1 into minor ginsenosides [22]. Although the enzymatic conversion of ginsenosides has been reviewed previously, including the conversion methods of ginsenosides [23], classification of ginsenosidases [22] and conversion of multiple ginsenosides by multiple glycosidases [17], the progress of research on the conversion of ginsenoside Rb1 by β-glucosidases has not been focused on.

2. Classification of Ginsenosidases

Ginsenosidases are commonly classified as type I, type II, type III and type IV, according to the hydrolysis site, residues and types of sugar moieties of ginsenosides. In addition, Shin et al., introduced the ginsenosidase type V to classify other ginsenosidases that had previously not been assigned [22]. Ginsenosidase type I simultaneously hydrolyze the glycosyl residues linked to C-20 and C-3 positions in the PPD-type ginsenosides, yielding minor ginsenosides with only one glucose residue or other glycosyl residues, such as ginsenosides CK and Rh2. The β-glucosidase from Microbacterium esteraromaticum, which belongs to ginsenosidase type I, hydrolyzes the C-3 glycosyl moieties of ginsenoside Rb1 as well as the C-20 glycosyl moieties to produce minor ginsenoside CK [24]. Ginsenosidase type II hydrolyzes the glycosyl residues at the C-20 position of PPD-and PPT-type saponins. Ginsenosidase from Aspergillus sp. g48p hydrolyses glycosyl residues at the C-20 of PPD-type ginsenosides (e.g., Rb1, Rb2 and Rc) into ginsenoside Rd and a small amount of ginsenoside Rg3, without hydrolysis of the glycosyl moieties at C-3 [25]. Ginsenosidase type III hydrolyzes the sugar moieties attached to the C-3 position of PPD-type ginsenosides, such as the enzyme from Terrabacter ginsenosidimutans, which hydrolyzes 3-O-β-D-(1-2)-glucopyranoside residue in Rb1 to produce gypenoside XVII (Gyp17), and then further hydrolyzes 3-O-β-D-glucopyranoside residue in Gyp17 to produce the end product gypenoside LXXV (Gyp75) [26]. Ginsenosidase type IV only hydrolyzes the sugar moieties linked to C-6 in PPT-type ginsenosides. For example, the enzyme from Aspergillus sp. strain 39 g hydrolyzes the glycosyl moieties attached to C-6 of ginsenosides Re and R1 to convert them into F1, and hydrolyzes the glycosyl moieties of ginsenoside Rg2 to produce Rh1, with no hydrolysis of PPD-type ginsenosides [27]. Ginsenosidase type V hydrolyzes the glycosyl moieties attached to the C-20 and C-6 positions of PPT-type ginsenosides to yield the corresponding aglycones. For example, the recombinant β-glycosidase from Actinosynnema mirum hydrolyzes the outer and inner glucose moieties linked to C-20 and C-6 of ginsenoside Rg1 to produce APPT [28].

References

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  26. Jin, X.F.; Yu, H.S.; Wang, D.M.; Liu, T.Q.; Liu, C.Y.; An, D.S.; Im, W.T.; Kim, S.G.; Jin, F.X. Kinetics of a cloned special ginsenosidase hydrolyzing 3-O-glucoside of multi-protopanaxadiol-type ginsenosides, named ginsenosidase type III. J. Microbiol. Biotechnol. 2012, 22, 343–351.
  27. Wang, D.M.; Yu, H.S.; Song, J.G.; Xu, Y.F.; Liu, C.Y.; Jin, F.X. A novel ginsenosidase from an Aspergillus strain hydrolyzing 6-O-multi-glycosides of protopanaxatriol-type ginsenosides, named ginsenosidase type IV. J. Microbiol. Biotechnol. 2011, 21, 1057–1063.
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Subjects: Biotechnology & Applied Microbiology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Hongrong Zhu
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Rui Zhang
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Zunxi Huang
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Junpei Zhou
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Revisions: 3 times (View History)
Update Date: 19 Jan 2023
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