scholarly journals A New Triterpenoid Glucoside from a Novel Acidic Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3457 ◽  
Author(s):  
Te-Sheng Chang ◽  
Chien-Min Chiang ◽  
Yu-Han Kao ◽  
Jiumn-Yih Wu ◽  
Yu-Wei Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Performance ◽  
Culture Collection ◽  
Subtilis Strain ◽  
Resonance Spectroscopy ◽  
Ganoderic Acid ◽  
Acidic Solutions ◽  
Medicinal Fungus ◽  
Bacillus Subtilis Atcc ◽  
Acidic Conditions

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-β-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-β-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-β-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min−1) was 3-fold higher than that at pH 7 (kcat = 3.8 min−1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.

scholarly journals Uridine Diphosphate-Dependent Glycosyltransferases from Bacillus subtilis ATCC 6633 Catalyze the 15-O-Glycosylation of Ganoderic Acid A

2018 ◽  
Vol 19 (11) ◽  
pp. 3469 ◽  
Author(s):  
Te-Sheng Chang ◽  
Jiumn-Yih Wu ◽  
Tzi-Yuan Wang ◽  
Kun-Yuan Wu ◽  
Chien-Min Chiang
Keyword(s):  
Bacillus Subtilis ◽  
Liquid Chromatography ◽  
High Performance ◽  
Ultra Performance Liquid Chromatography ◽  
Culture Collection ◽  
Resonance Spectroscopy ◽  
Uridine Diphosphate ◽  
Ganoderic Acid ◽  
Subtilis Atcc ◽  
Bacillus Subtilis Atcc

Bacillus subtilis ATCC (American type culture collection) 6633 was found to biotransform ganoderic acid A (GAA), which is a major lanostane triterpenoid from the medicinal fungus Ganoderma lucidum. Five glycosyltransferase family 1 (GT1) genes of this bacterium, including two uridine diphosphate-dependent glycosyltransferase (UGT) genes, BsUGT398 and BsUGT489, were cloned and overexpressed in Escherichia coli. Ultra-performance liquid chromatography confirmed the two purified UGT proteins biotransform ganoderic acid A into a metabolite, while the other three purified GT1 proteins cannot biotransform GAA. The optimal enzyme activities of BsUGT398 and BsUGT489 were at pH 8.0 with 10 mM of magnesium or calcium ion. In addition, no candidates showed biotransformation activity toward antcin K, which is a major ergostane triterpenoid from the fruiting bodies of Antrodia cinnamomea. One biotransformed metabolite from each BsUGT enzyme was then isolated with preparative high-performance liquid chromatography. The isolated metabolite from each BsUGT was identified as ganoderic acid A-15-O-β-glucoside by mass and nuclear magnetic resonance spectroscopy. The two BsUGTs in the present study are the first identified enzymes that catalyze the 15-O-glycosylation of triterpenoids.

scholarly journals Glycosylation of Ganoderic Acid G by Bacillus Glycosyltransferases

2021 ◽  
Vol 22 (18) ◽  
pp. 9744
Author(s):  
Jiumn-Yih Wu ◽  
Hsiou-Yu Ding ◽  
Tzi-Yuan Wang ◽  
Yun-Rong Zhang ◽  
Te-Sheng Chang
Keyword(s):  
Bacillus Subtilis ◽  
Ganoderma Lucidum ◽  
Aqueous Solubility ◽  
Triterpenoid Saponin ◽  
Bioactive Components ◽  
Ganoderic Acid ◽  
Triterpenoid Saponins ◽  
Medicinal Fungus ◽  
Bacillus Subtilis Atcc ◽  
New Compounds

Ganoderma lucidum is a medicinal fungus abundant in triterpenoids, its primary bioactive components. Although numerous Ganoderma triterpenoids have already been identified, rare Ganoderma triterpenoid saponins were recently discovered. To create novel Ganoderma saponins, ganoderic acid G (GAG) was selected for biotransformation using four Bacillus glycosyltransferases (GTs) including BtGT_16345 from the Bacillus thuringiensis GA A07 strain and three GTs (BsGT110, BsUGT398, and BsUGT489) from the Bacillus subtilis ATCC 6633 strain. The results showed that BsUGT489 catalyzed the glycosylation of GAG to GAG-3-o-β-glucoside, while BsGT110 catalyzed the glycosylation of GAG to GAG-26-o-β-glucoside, which showed 54-fold and 97-fold greater aqueous solubility than that of GAG, respectively. To our knowledge, these two GAG saponins are new compounds. The glycosylation specificity of the four Bacillus GTs highlights the possibility of novel Ganoderma triterpenoid saponin production in the future.

scholarly journals Production of New Isoflavone Glucosides from Glycosylation of 8-Hydroxydaidzein by Glycosyltransferase from Bacillus subtilis ATCC 6633

Catalysts ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 387 ◽  
Author(s):  
Chien-Min Chiang ◽  
Tzi-Yuan Wang ◽  
Szu-Yi Yang ◽  
Jiumn-Yih Wu ◽  
Te-Sheng Chang
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Nuclear Magnetic Resonance ◽  
Bacillus Subtilis ◽  
High Performance ◽  
Aqueous Solubility ◽  
Resonance Spectroscopy ◽  
Initial Concentration ◽  
Subtilis Atcc ◽  
Bacillus Subtilis Atcc

8-Hydroxydaidzein (8-OHDe) has been proven to possess some important bioactivities; however, the low aqueous solubility and stability of 8-OHDe limit its pharmaceutical and cosmeceutical applications. The present study focuses on glycosylation of 8-OHDe to improve its drawbacks in solubility and stability. According to the results of phylogenetic analysis with several identified flavonoid-catalyzing glycosyltransferases (GTs), three glycosyltransferase genes (BsGT110, BsGT292 and BsGT296) from the genome of the Bacillus subtilis ATCC 6633 strain were cloned and expressed in Escherichia coli. The three BsGTs were then purified and the glycosylation activity determined toward 8-OHDe. The results showed that only BsGT110 possesses glycosylation activity. The glycosylated metabolites were then isolated with preparative high-performance liquid chromatography and identified as two new isoflavone glucosides, 8-OHDe-7-O-β-glucoside and8-OHDe-8-O-β-glucoside, whose identity was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. The aqueous solubility of 8-OHDe-7-O-β-glucoside and 8-OHDe-8-O-β-glucoside is 9.0- and 4.9-fold, respectively, higher than that of 8-OHDe. Moreover, more than 90% of the initial concentration of the two 8-OHDe glucoside derivatives remained after 96 h of incubation in 50 mM of Tris buffer at pH 8.0. In contrast, the concentration of 8-OHDe decreased to 0.8% of the initial concentration after 96 h of incubation. The two new isoflavone glucosides might have potential in pharmaceutical and cosmeceutical applications.

scholarly journals One-Pot Bi-Enzymatic Cascade Synthesis of Novel Ganoderma Triterpenoid Saponins

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 580
Author(s):  
Te-Sheng Chang ◽  
Chien-Min Chiang ◽  
Tzi-Yuan Wang ◽  
Yu-Li Tsai ◽  
Yu-Wei Wu ◽  
...  
Keyword(s):  
High Performance ◽  
Aqueous Solubility ◽  
Major Product ◽  
Cyclodextrin Glucanotransferase ◽  
Bioactive Constituents ◽  
Ganoderic Acid ◽  
One Pot ◽  
Triterpenoid Saponins ◽  
Medicinal Fungus ◽  
Triterpenoid Glycosides

Ganoderma lucidum is a medicinal fungus whose numerous triterpenoids are its main bioactive constituents. Although hundreds of Ganoderma triterpenoids have been identified, Ganoderma triterpenoid glycosides, also named triterpenoid saponins, have been rarely found. Ganoderic acid A (GAA), a major Ganoderma triterpenoid, was synthetically cascaded to form GAA-15-O-β-glucopyranoside (GAA-15-G) by glycosyltransferase (BtGT_16345) from Bacillus thuringiensis GA A07 and subsequently biotransformed into a series of GAA glucosides by cyclodextrin glucanotransferase (Toruzyme® 3.0 L) from Thermoanaerobacter sp. The optimal reaction conditions for the second-step biotransformation of GAA-15-G were found to be 20% of maltose; pH 5; 60 °C. A series of GAA glucosides (GAA-G2, GAA-G3, and GAA-G4) could be purified with preparative high-performance liquid chromatography (HPLC) and identified by mass and nucleic magnetic resonance (NMR) spectral analysis. The major product, GAA-15-O-[α-glucopyranosyl-(1→4)-β-glucopyranoside] (GAA-G2), showed over 4554-fold higher aqueous solubility than GAA. The present study demonstrated that multiple Ganoderma triterpenoid saponins could be produced by sequential actions of BtGT_16345 and Toruzyme®, and the synthetic strategy that we proposed might be applied to many other Ganoderma triterpenoids to produce numerous novel Ganoderma triterpenoid saponins in the future.

scholarly journals Biotransformation of Ganoderic Acid A to 3-O-Acetyl Ganoderic Acid A by Soil-isolated Streptomyces sp.

Fermentation ◽  
2018 ◽  
Vol 4 (4) ◽  
pp. 101
Author(s):  
Te-Sheng Chang ◽  
Horng-Huey Ko ◽  
Tzi-Yuan Wang ◽  
Chun-Hsien Lee ◽  
Jiumn-Yih Wu
Keyword(s):  
Liquid Chromatography ◽  
High Performance ◽  
Ultra Performance Liquid Chromatography ◽  
Rrna Gene ◽  
Ganoderic Acid ◽  
Chromatography Method ◽  
Streptomyces Sp ◽  
Chromatography Analysis ◽  
Medicinal Fungus ◽  
Liquid Chromatography Method

The medicinal fungus Ganoderma lucidum contains many bioactive triterpenoids, ganoderic acid A (GAA) being one of the major ones. The present study explored the microbial biotransformation of GAA, isolating 283 strains of soil actinomycetes and determining their abilities to biotransform GAA with ultra-performance liquid chromatography analysis. One positive strain, AI 045, was selected to validate the biotransformation activity. The strain was identified as Streptomyces sp. based on the sequenced 16S rRNA gene. The produced compound obtained from the biotransformation of GAA was purified with the preparative high-performance liquid chromatography method and identified as 3-O-acetyl GAA based on mass and nuclear magnetic resonance spectral data. The present study is the first report that bacteria have the novel ability to biotransform the triterpenoids of fungus G. lucidum. Moreover, the identified 3-O-acetyl GAA is a new triterpenoid product discovered in microbes.

scholarly journals Analysis of the efficiency factors of electrotransformation of Bacillus subtilis to inactivate the aroK gene by the method of homologous recombination

2021 ◽  
pp. 64-73
Author(s):  
Yui Chao ◽  
Aleksei V. Lahodzich
Keyword(s):  
Bacillus Subtilis ◽  
Plasmid Dna ◽  
High Performance ◽  
Shikimic Acid ◽  
Fermentation Medium ◽  
Subtilis Strain ◽  
Culture Solution ◽  
Competent Cells ◽  
Integrative Plasmid ◽  
Efficiency Factors

A hyper-osmotic electrotransformation method was developed for strain Bacillus subtilis. Sorbitol and mannitol are included in the hyper-osmotic electroporation medium and recovery medium. In this study, the hyper-osmotic electroporation method was optimised to increase the transformation efficiency of B. subtilis strain 5434 (non-transformable by chemical methods) by 430 fold, with a maximum value of 8.6 ⋅ 105 CFU/µg of integrative plasmid DNA. With the electroporation setted 25 µF, 23 kV/cm, 200 Ω, the method was optimised as follows: a) the OD600 value of the bacterial culture solution was increased to about 1.2, which significantly enhanced survival of bacteria and quantity of viable B.subtilis strain 5434 cells after electroporation; b) the elution frequency of washing solution (hyper-osmotic electroporation medium) for complement cells was increased from 3 to 5 times, resulted in significantly reducing the conductivity of the hyper-osmotic electoporation medium with competent cells (electrocompetent cultue), and effectively extending the pulse time under the same electric field strength; c) quantity of integrative plasmid DNA added to hyper-osmotic electrocompetent culture was optimised. These results indicate that increasing the number of viable B. subtilis strain 5434 cells and reducing the number of metal ions in the electroporation solution mix (integrative plasmid DNA, competent cells of B. subtilis strain 5434, electroporation medium) are useful approach to improve transfomation efficiency of B. subtilis strain 5434. Concentration of shikimic acid in the fermentation medium was quantified by high performance liquid chromatography. Quantification of shikimic acid revealed that B. subtilis strain 5434p4SA produced 403.98 ± 9.1 µg/mL of shikimic acid.

scholarly journals Sequential Biotransformation of Antcin K by Bacillus subtilis ATCC 6633

Catalysts ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 349 ◽  
Author(s):  
Te-Sheng Chang ◽  
Chien-Min Chiang ◽  
Yi-Yun Siao ◽  
Jiumn-Yih Wu
Keyword(s):  
Nuclear Magnetic Resonance ◽  
High Performance Liquid Chromatography ◽  
Bacillus Subtilis ◽  
High Performance ◽  
Fruiting Bodies ◽  
Subtilis Atcc ◽  
Bacillus Subtilis Atcc ◽  
Triterpenoid Glycosides ◽  
New Compounds ◽  
First Time

The biotransformation of antcin K, a major ergostane triterpenoid from the fruiting bodies of Antrodia cinnamomea, by Bacillus subtilis (B. subtilis) ATCC 6633 was studied. Four metabolites from the biotransformation were isolated with preparative high-performance liquid chromatography and identified as 25S-antcin K 26-O-β-glucoside, 25R-antcin K 26-O-β-glucoside, 25S-antcin K 26-O-β-(6′-O-succinyl)-glucoside, and 25R-antcin K 26-O-β-(6′-O-succinyl)-glucoside with mass and nuclear magnetic resonance spectral analysis. By using either 25S-antcin K 26-O-β-glucoside or 25R-antcin K 26-O-β-glucoside as the biotransformation precursor, it was proven that 25S-antcin K 26-O-β-(6′-O-succinyl)-glucoside and 25R-antcin K 26-O-β-(6′-O-succinyl)-glucoside were biotransformed from 25S-antcin K 26-O-β-glucoside and 25R-antcin K 26-O-β-glucoside, respectively. To the best of our knowledge, this is the first study on the glycosylation of triterpenoids from A. cinnamomea, and the first time the succinylation of triterpenoid glycosides by microorganisms has been found. In addition, all four antcin K glucoside derivatives are new compounds.

Superionic Lithium Intercalation through 2 nm x 2 nm Columns in the Crystallographic Shear Phase Nb18W8O69

2019 ◽  
Author(s):  
Kent Griffith ◽  
Clare Grey
Keyword(s):  
High Performance ◽  
Rate Capability ◽  
Block Size ◽  
Lithium Intercalation ◽  
Resonance Spectroscopy ◽  
Complex Metal Oxides ◽  
Battery Electrode ◽  
Crystallographic Shear ◽  
Battery Material ◽  
First Time

Nb18W8O69 (9Nb2O5×8WO3) is the tungsten-rich end-member of the Wadsley–Roth crystallographic shear (cs) structures within the Nb2O5–WO3 series. It has the largest block size of any known, stable Wadsley–Roth phase, comprising 5 ´ 5 units of corner-shared MO6 octahedra between the shear planes, giving rise to 2 nm ´ 2 nm blocks. Rapid lithium intercalation is observed in this new candidate battery material and 7Li pulsed field gradient nuclear magnetic resonance spectroscopy – measured in a battery electrode for the first time at room temperature – reveals superionic lithium conductivity. In addition to its promising rate capability, Nb18W8O69 adds a piece to the larger picture of our understanding of high-performance Wadsley–Roth complex metal oxides.

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