• Applied Biological Chemistry
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• v.40 no.6
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• pp.465-471
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• 1997

The cyclodextrin glycosyltransferase (CGTase, EC 3.2.1.19) from Bacillus brevis CD162 was purified by precipitating with ammonium sulfate, DEAE-Sepharose CL-6B column chromatography and Sephadex G-150 column chromatography. The molecular mass and pI of the purified enzyme were estimated to be 74,000 and 6.3 by SDS-PAGE and isoelectric focusing, respectively. The purified enzyme was clearly identified as the CGTase by zymogram after SDS-PAGE. The optimum pH and temperature for the enzyme activity were 8.0 and $55^{\circ}C$, respectively. The enzyme was stable at the range of pH $5.5{\sim}9.0$, and up to $50^{\circ}C$. The amino acid sequence from the $NH_2-terminal$ of the purified CGTase was Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln. The yields of the products from starch as the substrate were 1.3% for ${\alpha}-$, 33.9% for ${\beta}-$, and 9.7% for ${\gamma}-cyclodextrin$.

• Applied Biological Chemistry
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• v.40 no.6
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• pp.459-464
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• 1997

A cyclodextrin glycosyltransferase-producing bacterium was newly isolated from soil using alkaline pH medium containing 1% $Na_2CO_3$. The isolated strain was identified as Bacillus brevis by morphological and biochemical characteristics, and fatty acid composition and designated Bacillus brevis CD162. The strain showed the best enzyme production of 0.9 unit/ml after 96 hrs of culture at $30^{\circ}C$ in a medium of 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% $K_2HPO_4$ 0.05% $MgSO_4{\cdot}7H_2O$, and 1.5% $Na_2CO_3$ at initial pH 10.2.

• Journal of Microbiology and Biotechnology
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• v.17 no.3
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• pp.539-542
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• 2007

Glycosyltransferase family 1 (UOT) uses small chemicals including phenolics, antibiotics, and alkaloids as substrates to have an influence in biological activities. A glycosyltransferase (XcGT-2) from Xanthomonas campestris was cloned and consisted of a 1,257 bp open reading frame encoding a 45.5 kDa protein. In order to use this for the modification of phenolic compounds, XcGT-2 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. With the E. coli transformant expressing XcGT-2, biotransformation of flavonoids was carried out. Flavonoids having a double bond between carbons 2 and 3, and hydroxyl groups at both C-3' and C-4', were glycosylated and the glycosylation position was determined to be at the hydroxyl group of C-3', using nuclear magnetic resonance spectroscopy. These results showed that XcGT-2 regiospecifically transferred a glucose molecule to the 3'-hydroxyl group of flavonoids containing both 3' and 4'-hydroxyl groups.

• Korean Journal of Food Science and Technology
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• v.25 no.6
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• pp.608-613
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• 1993

Production of cyclodextrin (CD) by cyclodextrin glycosyltransferase (CGTase) isolated from alkalophilic Bacillus sp. was carried out to determine optimal reaction conditions. The maximum initial rate of CD production from amylose was obtained at dextrose equivalent 10.5. The CD production yield showed inverse proportionality to DE values over the range from 0.5 to 37.7. Even though the deactivation constant of CGTase at $60^{\circ}C$ was higher than those at lower temperatures, the production rate and yield at $60^{\circ}C$ were still higher. These results suggest thermal stabilization of CGTase by binding with starch.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.657-662
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• 2022

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli, and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α-O-glucoside and phenoxodiol-7-α-O-glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.

• KSBB Journal
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• v.26 no.2
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• pp.93-99
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• 2011

Glycosylation is a key mechanism in determining diversity of natural products, and influencing their bioactivities. This approach requires a core set of glycosyltransferase that synthesizes the diverse sugar structures observed in nature. Recently, the researchers have begun to alter the sugar moiety and glycosylation patterns of natural products both in vivo E. coli system and in vitro for their glycodiversification. This review highlights new glycosylation tools using microbialproduced deoxysugar and a flexible glycosyltransferase on natural plant-flavonoids to generate novel glycoforms with useful biological activity.

• Journal of Ginseng Research
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• v.42 no.1
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• pp.42-49
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• 2018

Background: Ginsenoside F1 has been described to possess skin-whitening effects on humans. We aimed to synthesize a new ginsenoside derivative from F1 and investigate its cytotoxicity and melanogenesis inhibitory activity in B16BL6 cells using recombinant glycosyltransferase enzyme. Glycosylation has the advantage of synthesizing rare chemical compounds from common compounds with great ease. Methods: UDP-glycosyltransferase (BSGT1) gene from Bacillus subtilis was selected for cloning. The recombinant glycosyltransferase enzyme was purified, characterized, and utilized to enzymatically transform F1 into its derivative. The new product was characterized by NMR techniques and evaluated by MTT, melanin count, and tyrosinase inhibition assay. Results: The new derivative was identified as (20S)-$3{\beta},6{\alpha},12{\beta}$,20-tetrahydroxydammar-24-ene-20-O-${\beta}$-D-glucopyranosyl-3-O-${\beta}$-D-glucopyranoside(ginsenoside Ia), which possesses an additional glucose linked into the C-3 position of substrate F1. Ia had been previously reported; however, no in vitro biological activity was further examined. This study focused on the mass production of arduous ginsenoside Ia from accessible F1 and its inhibitory effect of melanogenesis in B16BL6 cells. Ia showed greater inhibition of melanin and tyrosinase at $100{\mu}mol/L$ than F1 and arbutin. These results suggested that Ia decreased cellular melanin synthesis in B16BL6 cells through downregulation of tyrosinase activity. Conclusion: To our knowledge, this is the first study to report on the mass production of rare ginsenoside Ia from F1 using recombinant UDP-glycosyltransferase isolated from B. subtillis and its superior melanogenesis inhibitory activity in B16BL6 cells as compared to its precursor. In brief, ginsenoside Ia can be applied for further study in cosmetics.

• Korean Journal of Food Science and Technology
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• v.26 no.4
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• pp.375-381
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• 1994

A bacterial strain No. KY-126, which produced extracellular cyclodextrin glycosyltransferase(CGTase), was isolated from soil and identified as Bacillus stearothermophilus KY-126. The enzyme was purified by the treatments of ammonium sulfate precipitation, DEAF-Sephadex, Sephadex G-100 column chromatography. The optimal pH and temperature for the enzyme activity were pH 5.5 and $65^{\circ}C$, respectively. And the enzyme was stable at pH values from 6.0 to 11.0 at $55^{\circ}C$ for 30 min and stable up to $60^{\circ}C$ for 30 min.. The enzyme was inhibited by $HgCl_{2}$. The molecular weight of the enzyme was estimated to be 67,000 by using SDS-PAGE. The maximum conversion from starch to cyclodextrin (CD) by CGTase was 43% and obtained at 6 hr reaction and the ratio of ${\alpha}-,\;{\beta}-,\;{\gamma}-$, CD production at this time was 2.9 : 2.1 : 1.0.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.725-729
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• 2008

An enzyme reactor installed with ultrafiltration membrane was developed to produce ${\alpha}-,\;{\beta}-$, and ${\gamma}$-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTKIOase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a ${\alpha}$-CD yield of 24.0 (${\pm}1.5$)% and a productivity of 4.68 (${\pm}0.88$) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1-nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.

• Molecules and Cells
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• v.27 no.1
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• pp.83-88
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• 2009

Amino acid homology analysis predicted that rbmD, a putative glycosyltransferase from Streptomyces ribosidificus ATCC 21294, has the highest homology with neoD in neomycin biosynthesis. S. fradiae BS1, in which the production of neomycin was abolished, was generated by disruption of the neoD gene in the neomycin producer S. fradiae. The restoration of neomycin by self complementation suggested that there was no polar effect in the mutant. In addition, S. fradiae BS6 was created with complementation by rbmD in S. fradiae BS1, and secondary metabolite analysis by ESI/MS, LC/MS and MS/MS showed the restoration of neomycin production in S. fradiae BS6. These gene inactivation and complementation studies suggested that, like neoD, rbmD functions as a 2-N-acetlyglucosaminyltransferase and demonstrated the potential for the generation of novel aminoglycoside antibiotics using glycosyltransferases in vivo.