• Applied Biological Chemistry
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• 제40권6호
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• pp.465-471
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• 1997

Bacillus brevis CD162가 생산하는 cyclodextrin glycosyltransferase (CGTase)를 ammonium sulfate 침전, DEAE-Sephadex CL-6B 및 Sephadex G-150 컬럼 크로마토그래피를 수행하여 분리정제하였다. 정제된 CGTase는 분자량이 약 74,000, 등전점은 약 6.3인 단백질이었고, 정제된 단백질을 SDS-PAGE한 후 변성된 단백질을 재활성시켜 zymogram을 수행한 결과 cyclodextrin glycosyltransferase임을 확인할 수 있었다. 이 효소의 최적활성 pH와 온도는 각각 8.0과 $55^{\circ}C$이었으며, pH $5.5{\sim}9.0$과 $50^{\circ}C$까지 안정한 활성을 보였다. 또한, CGTase의 $NH_2$-말단 부위의 아미노산서열은 Ser-Val-Thr-Asn-Lys-Val-Asn-Tyr-Ser-Lys-Asp-Val-Ile-Tyr-Gln 이었으며, 전분으로부터 cyclodextrin으로의 전환률을 분석한 결과, ${\alpha}$-cyclodextrin은 1.3%, ${\beta}$-cyclodextrin은 33.9% ${\gamma}$-cyclodextrin은 9.7% 이었다.

• Applied Biological Chemistry
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• 제40권6호
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• pp.459-464
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• 1997

Cyclodextrin glycosyltransferase 생산균주를 토양으로부터 분리하여 형태학적, 생화학적 그리고 균주의 세포벽 지방산 조성분석에 의해 Bacillus brevis로 동정하였고, Bacillus brevis CD162로 명명하였다. 또한 배지조성에 따른 cyclodextrin glycosyltransferase의 최적생산조건을 검토한 결과, 2.0% soluble starch, 0.75% yeast extract, 0.5% bacto peptone, 0.2% $K_2HPO_4$, 0.05% $MgSO_4{\cdot}7H_2O$, 1.5% $Na_2CO_3$ (pH 10.2)의 배지 조건에서 $30^{\circ}C$에서 96시간 배양하였을 때 가장 높은 효소생산인 0.9 unit/ml을 얻을 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제17권3호
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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.

• 한국식품과학회지
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• 제25권6호
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• pp.608-613
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• 1993

본 실험에서는 토양에서 분리한 호알카리성 Bacillus sp. 유래의 cyclodextrin glycosyltransferase(CGTase)를 이용하여 전분을 기질로 하였을 때 CD의 생산에 미치는 여러 조건을 고찰하였다. CD의 초기생성속도를 최대로 하는 기질의 최적 dextrose equivalent(DE)값은 10.5이었고 이로부터 CD 생산 반응인 cyclization에 필요한 기질의 최적 DE값이 존재함을 알았다. 온도가 증가할수록 CGTase의 활성도는 급격히 감소하였지만 CD 생산속도와 수율은 온도에 따라 증가하였다. 이는 CD 생산반응중 CGTase는 기실과 결합하여 열에 대해 안정성이 증가하기 때문인 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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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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• 제26권2호
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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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• 제42권1호
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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.

• 한국식품과학회지
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• 제26권4호
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• pp.375-381
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• 1994

토양을 대상으로 하여 CGTase를 생산하는 균주를 분리, 선별하여 Bacillus stearothermophilus KY-126을 얻었다. CGTase의 정제는 ammonium sulfate precipitation, ion exchange chromatography, gel filtration의 과정을 통해 분리 정제하여 단일 효소를 얻었으며, 분자량은 약 67,000이었다. 효소 반응의 최적 온도는 $65^{\circ}C$였으며, $55^{\circ}C$에서 30분간 열처리에도 비교적 열에 안정하였다. 최저 활성 pH는 5.5였고 pH5.5에서 10.5까지 비교적 안정하였다. $HgCl_{2}$에 의해 저해를 받았으며, 그 외의 금속 이온에는 저해를 받지 않았다. Soluble starch로부터 CD의 전환율은 43%이었으며, ${\alpha}-:,\;{\beta}-:,\;{\gamma}-$, CD의 생성 비율은 2.9 : 2.1 : 1이었다.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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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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• 제27권1호
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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.