Highly Selective Production of Compound K from Ginsenoside Rd by Hydrolyzing Glucose at C-3 Glycoside Using ��-Glucosidase of Bifidobacterium breve ATCC 15700

Zhang, Ru; Huang, Xuemei; Yan, Hui‐Juan; Liu, Xin-Yi; Zhou, Qi; Luo, Zhiyong; Tan, Xiaoning; Zhang, Bian-Ling

doi:10.4014/jmb.1808.08059

Cited by 30 publications

(17 citation statements)

References 26 publications

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“…The kinetic parameters of BsAbfA were measured using p NP-Af and Rc as substrate at concentrations ranging from 0.1 to 5 mM. The K m , K cat , and V max were calculated by fitting the activity data to a linear regression on Lineweaver–Burk double-reciprocal plots [ 47 ]. The substrate specificity of purified recombinant BsAbfA was assayed by using p NP-α-Af, p NP-α-Ap, p NP-α-Rp, p NP-β-Glc, ginsenoside Rb 1 , Rb 2 , Rc, Rd, Re, Rg 1 , F 2 , C-K, C-Mc 1 , C-Mc, gentiobiose, and sophorose as substrates, individually.…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was removed by a magnetic separator and washed at least twice with elution buffer. Purified proteins were maintained in 50 mM citric acid/sodium citrate buffer (pH 5) at 4 • C. The concentration of purified recombinant BsAbfA was assayed using Folin-phenol reagent [47]. The expression quantity and molecular weight of the protein were analyzed by SDS-PAGE.…”

Section: Cloning Site-directed Mutagenesis Heterologous Expression and Protein Purificationmentioning

confidence: 99%

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Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

et al. 2021

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α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other β-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters Km of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the Kcat/Km were 181.5 s−1 mM−1 and 197.8 s−1 mM−1, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C20 position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.

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Section: Methodsmentioning

confidence: 99%

Section: Cloning Site-directed Mutagenesis Heterologous Expression and Protein Purificationmentioning

confidence: 99%

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

et al. 2021

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“…Apart from commercial enzymes used for hydrolysis of ginsenosides, enzymes isolated from bacteria and fungi have also been recombined in host strains to enhance the enzyme activity. Several recombinant β-glucosidases show outstanding abilities for conversion of Rg3, Rh2, and CK by hydrolysis of the glucose at C–3 and C–20 [ 119 , 120 , 121 ]. Recombinant β-glucosidase (bgp 1) not only transforms Rb1 and Rd into Rg3, but also generates Rg2 and Rh1 from Re when reacted with ginseng leaf saponins [ 122 ].…”

Section: Variations In Ginsenoside Compositions Due To Different Pmentioning

confidence: 99%

Diversity of Ginsenoside Profiles Produced by Various Processing Technologies

et al. 2020

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Ginseng is a traditional medicinal herb commonly consumed world-wide owing to its unique family of saponins called ginsenosides. The absorption and bioavailability of ginsenosides mainly depend on an individual’s gastrointestinal bioconversion abilities. There is a need to improve ginseng processing to predictably increase the pharmacologically active of ginsenosides. Various types of ginseng, such as fresh, white, steamed, acid-processed, and fermented ginsengs, are available. The various ginseng processing methods produce a range ginsenoside compositions with diverse pharmacological properties. This review is intended to summarize the properties of the ginsenosides found in different Panax species as well as the different processing methods. The sugar moiety attached to the C–3, C–6, or C–20 deglycosylated to produce minor ginsenosides, such as Rb1, Rb2, Rc, Rd→Rg3, F2, Rh2; Re, Rf→Rg1, Rg2, F1, Rh1. The malonyl-Rb1, Rb2, Rc, and Rd were demalonylated into ginsenoside Rb1, Rb2, Rc, and Rd by dehydration. Dehydration also produces minor ginsenosides such as Rg3→Rk1, Rg5, Rz1; Rh2→Rk2, Rh3; Rh1→Rh4, Rk3; Rg2→Rg6, F4; Rs3→Rs4, Rs5; Rf→Rg9, Rg10. Acetylation of several ginsenosides may generate acetylated ginsenosides Rg5, Rk1, Rh4, Rk3, Rs4, Rs5, Rs6, and Rs7. Acid processing methods produces Rh1→Rk3, Rh4; Rh2→Rk1, Rg5; Rg3→Rk2, Rh3; Re, Rf, Rg2→F1, Rh1, Rf2, Rf3, Rg6, F4, Rg9. Alkaline produces Rh16, Rh3, Rh1, F4, Rk1, ginsenoslaloside-I, 20(S)-ginsenoside-Rh1-60-acetate, 20(R)-ginsenoside Rh19, zingibroside-R1 through hydrolysis, hydration addition reactions, and dehydration. Moreover, biological processing of ginseng generates the minor ginsenosides of Rg3, F2, Rh2, CK, Rh1, Mc, compound O, compound Y through hydrolysis reactions, and synthetic ginsenosides Rd12 and Ia are produced through glycosylation. This review with respect to the properties of particular ginsenosides could serve to increase the utilization of ginseng in agricultural products, food, dietary supplements, health supplements, and medicines, and may also spur future development of novel highly functional ginseng products through a combination of various processing methods.

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“…To minimize processing time and production cost, recombinant enzymes obtained from E. coli strains are commonly applied for the ginsenoside conversion. For instance, the recombinant β-glucosidase from Bi dobacterium breve ATCC 15700 was identi ed to produce CK from ginsenoside Rd (Zhang et al 2019). Higher productivity was achieved when recombinant thermophilic enzymes were used to transform ginsenosides.…”

Section: Introductionmentioning

confidence: 99%

“…These ginsenoside-transforming glycosidases including β-glucosidases, arabinopyranosidases, arabinofuranosidases, xylosidases, rhamnosidases and βgalactosidases belong to GH family 1, 2, 3, 39, 42, 51, and 78 (Shin and Oh 2015). For instance, The GH1 β-glucosidase from Sphingopyxis alaskensis speci cally hydrolyzed the outer glucose at the C3 position in PPD-type ginsenosides (Zhang et al 2019). Thermophilic glycosidases from GH family 1, 3, 39 and 42 were also identi ed to transform PPD-or PPT-type ginsenosides Pei et al 2015;Xie et al 2015;Shin et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Cloning and characterization of thermophilic cellulase and its application in the transformation of ginsenosides

Zheng

Zhao

Wang

et al. 2022

Preprint

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A novel cellulase (BcelFp) was identified from Fervidobaterium pennivorans DSM9078 which had biotransformation activity for PPD-type ginsenosides. Sequence analysis of BcelFp revealed that it could be classified into glycoside hydrolase family 5 (GH5). The gene encoding a 323-amino acid protein was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximately 37 kDa. The recombinant BcelFp exhibited an optimal activity at 95 oC and pH 5.5 and showed high thermostability. The cellulase had high selectivity for cleaving the outer glucose moiety at the C3 carbon of ginsenoside Rb1, Rb2, Rc and Rd, which produced the more pharmacologically active gypenoside XVII (GypXVII), Compound O (CO), Compound Mc1 (CMc1) and F2, respectively. The Km values for Rb1, Rb2, Rc and Rd were 3.66 ± 0.04 µM, 4.02 ± 0.12 µM, 5.95 ± 0.03 µM, 0.67 ± 0.006 µM, respectively. The kcat/Km value of BcelFp for ginsenoside Rd was 27.91 mM-1s-1, which was much higher than the previously enzymes. This study was the first report of the highly efficient and selective transformation of GypXVII, CO, CMc1 and F2 from Rb1, Rb2, Rc and Rd by a GH5-family thermophilic cellulase.

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Highly Selective Production of Compound K from Ginsenoside Rd by Hydrolyzing Glucose at C-3 Glycoside Using ��-Glucosidase of Bifidobacterium breve ATCC 15700

Cited by 30 publications

References 26 publications

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

Diversity of Ginsenoside Profiles Produced by Various Processing Technologies

Cloning and characterization of thermophilic cellulase and its application in the transformation of ginsenosides

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