March 10, 2021 Longchang Chemical
At present, glycoside hydrolases are used in the study of the preparation of a variety of active glycosides and aglycons. Among them, the enzymatic preparation of saponin and flavonoid glycosides in oxyglycosides is the most extensive. After years of efforts by scientific researchers, glycoside hydrolase has achieved many gratifying results in the preparation of active glycosides and aglycones.

1. Biotransformation of flavonoid glycosides

Flavonoids are polyphenols widely distributed in plants, mostly in the form of glycosides. Studies have found that flavonoids with biological activity are the most important active ingredients in edible plants, and have multiple pharmacological activities such as liver protection, anti-oxidation, anti-tumor, and anti-virus, and their activity is closely related to structure. Since most of the flavonoid glycosides are difficult to enter the blood through the wall of the small intestine, and their bioavailability is low, structural modification of natural flavonoids has become a hot spot in current research. The use of glycoside hydrolase to hydrolyze the glycosyl groups of flavonoid glycosides has become an effective way to improve the activity of flavonoids (Table 1). Common flavonoid glycosides include rutin, hesperidin and naringin, and their sugar moieties are usually rutin (α-1,6 linked rhamnose and glucose) and new hesperose (α-1,2 Connected rhamnose and glucose), so glycoside hydrolase hydrolyzes the modification of it mainly includes two kinds of exo- and endo-excision. From Aspergillus niger and Aspergillus nidulans, α-rhamnosidase, which can hydrolyze α-1,2 and α-1,6 rhamnoside bonds, was isolated and purified. This enzyme can hydrolyze rutin, naringin and hesperidin, Respectively produce isoquercetin, plumoside and hesperetin glucoside. The recombinant α-rhamnosidase cloned from Aspergillus aculeatus and Clostridium stercorarium also has the activity of hydrolyzing rhamnose in flavonoid glycosides. For the hydrolysis of the above three flavonoid glycosides, in addition to exoglycosidases, endoglycosidases also have a large number of research reports. Diglycosidase isolated and purified from Penicillium rugulosum, Penicillium decumben[ and Fagopyri herba, as well as recombinant rutinase cloned from Aspergillus niger, can hydrolyze rutin to produce quercetin with better antioxidant activity. Naringinase can be isolated and purified from Aspergillus niger BCC 25166 which can hydrolyze naringin to produce naringin. The naringinase in Aspergillus niger 1344 can hydrolyze naringin and rutin at the same time to produce naringin respectively. Yuan and quercetin, but hesperidin cannot be hydrolyzed. The diglycosidase in Acremonium sp. DSM24697 and Actinoplanes missouriensis can hydrolyze the new hesperidin in hesperidin to produce highly active hesperetin products.

Table 1. Biotransformation of flavonoid glycosides by glycosidase

ProductSubstrateReactionOrganism
Isoquercitrin, Prunin,Rutin, Naringin,α-RhamnosidaseAspergillus niger
Hesperetin glucosideHesperidinα-RhamnosidaseAspergillus nidulans
  α-RhamnosidaseAspergillus aculeatus
  α-RhamnosidaseClostridium stercorarium
QuercetinRutinβ-RutinosidasePenicillium rugulosum
QuercetinRutinβ-GlycosidasePenicillium decumbens
QuercetinRutinβ-HeterodisaccharidaseFagopyri herba
QuercetinRutinβ-RutinosidaseAspergillus niger
NaringeninNaringinNaringinaseAspergillus niger
Naringenin, QuercetinNaringin, RutinNaringinaseAspergillus niger
HesperetinHesperidinDiglycosidaseAcremonium
HesperetinHesperidinDiglycosidaseActinoplanes missouriensis
DaidzeinDaidzinβ-GlucosidaseUnculturable microbes
DaidzeinDaidzinβ-GlucosidaseSulfolobus solfataricus
DaidzeinDaidzinβ-GlucosidaseAspergillus oryzae
DaidzeinDaidzinβ-GlucosidasePyrococcus furiosus
Daidzein, GenisteinDaidzin, Genistinβ-GlucosidaseBacillus subtilis
  β-GlucosidaseThermotoga maritima
Daidzein, Genistein,Daidzin, Genistin,β-GlucosidaseDalbergia
GlyciteinGlycitinβ-GlucosidaseBacteroides thetaiotaomicron
BaicaleinBaicalinβ-GlucuronidaseScutellaria viscidula
TilianinLinarinNaringinasePenicillium decumbens
ButinButrinβ-GlucosidaseAlmond
PhloretinPhlorizinβ-GlycosidaseSheep small intestine
Isoflavones are a kind of flavonoids, which are mainly found in legumes, which contribute to disease prevention and human health. The main components of soy isoflavones are daidzein, daidzein, genistin, genistein, glycitein and glycitein aglycone, among which deglycosylated aglycone has better biological activity. Recombinant β-glucosidase capable of hydrolyzing daidzein to produce daidzein was cloned from the gene library of mangrove soil, Sulfolobus solfataricus, Aspergillus oryzae and Pyrococcus furiosus; recombinant β-glucosidase cloned from Thermotoga maritima and Bacillus subtilis Enzymes can hydrolyze daidzein and genistein to produce daidzein and genistein; the glycosidase isolated and purified from Dalbergia and cloned and recombined from Bacteroides thetaiotaomicron can hydrolyze daidzein, genistin and daidzein to produce daidzein, Genistein and glycitein aglycone.
Glycoside hydrolase has also been reported and applied in other flavonoid glycoside hydrolysis (Table 1). Studies have shown that baicalin has anti-tumor and anti-infection effects. The β-glucosidase isolated and purified from Scutellaria viscidula Bge can hydrolyze baicalin to produce baicalein. The deglycosylated product, baicalein, has better pharmacological activity. Serimarin is also a rare flavonoid glycoside with antihypertensive and sedative activities, but it is difficult to obtain by direct extraction and chemical synthesis. Cui et al. used naringinase to hydrolyze the rhamnose in montanoside to produce serimarin. In addition, Jassbi et al. used β-glucosidase to hydrolyze butrin to produce butrin. The results of antioxidant experiments showed that deglycosylated butrin had better activity than butrin. Day and other glycosidases isolated and purified from the small intestine of sheep can hydrolyze phlorizin to produce phloretin.

2. Biotransformation of other oxyglycosides

In addition to saponins and flavonoid glycosides, glycoside hydrolases have also been used to hydrolyze and modify other oxygen glycosides (Table 2). Gardenia fruit is a traditional Chinese medicine used to treat cardiovascular, cerebrovascular, liver and gallbladder diseases. There are a lot of geniposide in gardenia fruits, but the effective ingredient is geniposide, which is the deglycosylated product of geniposide, and the content is less than 0.01%. The β-glucosidase isolated and purified from Penicillium nigricans and Aspergillus niger can be transformed into geniposide to prepare genipin to meet the demand for large quantities of genipin. Arctium lappa has the effects of preventing or treating chronic renal failure, and its effective ingredients are arctiin and arctigenin. The β-glucosidase in Grifola frondosa and Rhizoctonia solani can transform burdock to produce arctigenin. Liu et al. used commercial β-glucosidase to completely hydrolyze the burdock fruit to obtain the burdock aglycon product. The conversion of arctiin into arctigenin can effectively improve the bioavailability. Resveratrol has the functions of preventing tumors and atherosclerosis. The β-glucosidase isolated and purified from Aspergillus oryzae sp. 100 and Lactobacillus kimchi, and the recombinant β-glucosidase cloned from the metagenomics of mangrove soil by Mai et al. Glucosidase can convert polydatin to produce resveratrol. Paclitaxel is a secondary metabolite of Taxus chinensis plant and has a good therapeutic effect on ovarian cancer and breast cancer. The dry weight of paclitaxel in Taxus chinensis is only 0.02%, and the content of 7-xylose-10-deacetylpaclitaxel discarded as waste is more than 10 times that of paclitaxel. Dou et al. used the extracellular xylosidase secreted by Cellulosimicrobium cellulans strain F16 to convert 7-xylose-10-deacetylpaclitaxel into 10-deacetylpaclitaxel, and then through a one-step acylation reaction to generate paclitaxel.

Table 2. Biotransformation of other O-glycosides by glycosidase

ProductSubstrateReactionOrganism
GenipinGeniposideβ-GlucosidasePenicillium nigricans
GenipinGeniposideβ-GlucosidaseAspergillus niger
ArctigeninArctiinβ-GlucosidaseGrifola frondosa
ArctigeninArctiinβ-GlucosidaseRhizoctonia solani
ArctigeninArctiinβ-GlucosidaseCommercial
ResveratrolPolydatinβ-GlucosidaseAspergillus oryzae
ResveratrolPolydatinβ-GlucosidaseLactobacillus kimchi
ResveratrolPolydatinβ-GlucosidaseUnculturable microbes
10-Deacetylpaclitaxel7-Xylosyl-10-deacetylpaclitaxelβ-XylosidaseCellulosimicrobium cellulans

3. Biotransformation of carbon glycosides and thioglycosides

In addition to oxyglycosides, glycoside hydrolases are also used in the study of the hydrolysis modification of carbon glycosides and thioglycosides (Table 3). Carbon glycosides are formed by dehydration condensation of ortho- or para-position hydrogen activated by aglycone phenolic hydroxyl group with sugar groups. Carboside flavonoids have many activities such as anti-inflammatory, anti-bacterial, anti-tumor, lowering blood sugar and enhancing immunity. Compared with oxoside flavonoids, carboside flavonoids have higher stability and may be completely absorbed and become potential drug molecules. Since carbon glycosidic bonds are difficult to be hydrolyzed, there are few reports on the hydrolysis of carbon glycoside flavonoids. Sanugul et al. isolated a bacterium from a mixture of human fecal bacteria, which secreted a glycosidase under the induction of mangiferin, which can hydrolyze the carbon glycosidic bonds in mangiferin to produce mangiferin with better activity. Nakamura et al. isolated strain PUE from human intestinal bacteria, which can isolate and purify a carboglycosidase that hydrolyzes puerarin to produce aglycon. In the study of carboglycosidase-encoding genes, Braune et al. found that the protein-coding genes dfgA, dfgB, dfgC, dfgD and dfgE in Eubacterium cellulosolvens co-express carboglycosidases that can hydrolyze isoorientin to produce corresponding aglycons.
Glucosinolates are an important class of glucosinolate compounds, which are widely found in cruciferous plants, such as mustard, broccoli, garlic and so on. Studies have shown that eating cruciferous plants can effectively prevent breast cancer, lung cancer, colon cancer and other cancers. The main active ingredient is isothiocyanate produced after glucosinolate degradation. Glucosidase, also known as myrosinase, is mainly found in cruciferous plants, but it is distributed in a different position from glucosinolates. Only when the cells are broken, they will mix and react. Due to the low content of endogenous myrosinase, it is difficult to effectively hydrolyze glucosinolates to produce active products. Sulforaphane is an isothiocyanate with pharmacological activity. Shen et al. used exogenous myrosinase to successfully convert glucoraphanin into sulforaphane. At present, there are few studies on the hydrolytic modification of carbon glycosides and thioglycosides. In the future, the development and structural modification of more carbon glycosides and thioglycosides will provide more candidate molecules for drug development.

Table 3. Biotransformation of C-glycosides and S-glycosides by glycosidase.

ProductSubstrateReactionOrganism
NorathyriolMangiferinC-glucosyl-cleaving enzymeBacteroides
DaidzeinPuerarinC-glucosyl-cleaving enzymeHuman intestinal bacterium
LuteolinHomoorientinC-glucosyl-cleaving enzymeEubacterium cellulosolvens
SulforaphaneGlucoraphaninMyrosinaseBroccoli seeds

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