• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.402-406
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• 1996

Inulin fructotransferase (depolymerizing) (EC 2.4.1.93) was purified 34-fold from the culture broth of Arthrobacter sp. A-6 by using a combination of ammonium sulfate fractionation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The purified enzyme converts inulin into di-D-fructofuranose dianhydride III(DFA III) and small quantities of fructo-oligosaccharides. The temperature and pH optima of the enzyme were $70^{\circ}C$ and 6.0, respectively. Molecular weight of the enzyme was determined to be 49 kDa by 12$%$ SDS-polyacrylamide gel electrophoresis, and 145 kDa by Sephacryl S-200gel filtration. This indicates that the functional inulin fructotransferase of Arthrobacter sp. A-6 has a homomeric trimer structure. The enzyme had an isoelectric point of pH 4.6. The N-terminal amino acid sequence of the purified enzyme subunit was Ala-Asp-Asn-Pro-Asp-Gly(\ulcorner)-Ser-Asn-Met(or Glu)-Tyr-Asp-Val.

• 한국미생물·생명공학회지
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• 제23권1호
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• pp.68-74
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• 1995

A bacterial strain A-6 producing the high level of an extracellular inulin fructotransfe rase(depolymerizing)(EC 2.4.1.93) which converts inulin into di-D-fructofuranose dianhydride III (DFAIII) was isolated from soil. The isolated strain could be classified as a species belonging to the genus Arthrobacter based on its morphological and physiological characteristics identified in this work. Production of the enzyme was induced by inulin, and the highest activity was detected in the slightly acidic medium supplemented with 2.5% inulin and 0.1% trypton as a sole carbon and a nitrogen source, respectively. Under the optimal conditions, the enzyme activity in the culture supernatant reached approximately 60 uints/ml after 96 hours of cultivation. The optimum pH and temperature for the crude enzyme preparation from Arthrobacter sp. A-6 were pH 5.0 and 60$\circ$C , respectively. The DFA produced by the action of the inulin fructotransferase was confirmed to be DFAIII by paper chromatography, HPLC and $^{13}$C-NMR spectroscopy.

• 한국미생물·생명공학회지
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• 제21권1호
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• pp.36-40
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• 1993

토양에서 inulin으로부터 di-D-fructofuranose dianhydride(DFA)를 생산하는 inulin fructotransferase를 생산하는 균주로 Enterobacter sp. S45를 선발, 분리하였다. 본 효소에 의하여 생산되는 DFA는 $^13C-nmr$과 HPLC로 분석한 결과 fructose 두 분자가 $\beta$ 1,2': $\alpha$ 2,3' 결합을 한 DFA III로 동정되었다. 본 균주는 탄소원으로 inulin, 유기질소원으로 corn step liquor, 무기질소원으로 $NH_4H_2P0_4$를 사용할 때 효소생산이 가장 많았으며 이 조건하에서 72시간 배양으로 최대 효소생산을 보였다. 균 배양중 배양액내에 DFA III가 생성되었다가 소멸되는 것으로 보아 균체내에 DFAIII를 분해, 이용하는 효소가 존재한다고 추정되었다.

• Journal of Microbiology and Biotechnology
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• 제7권2호
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• pp.121-126
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• 1997

A bacterial strain LC-413, producing an extracellular inulin fructotransferase (depolymerizing) which converts inulin into di-D-fructofuranose dianhydride (DFAIII), was isolated from soil. Inulin fructotransferase from the isolate identified as a strain Flabobacterium sp. was purified from the culture broth by ammonium sulfate precipitation, followed by column chromatograpies on DEAE-Toyopearl 650 M and phenyl-Toyopearl 650 M. The purified enzyme gave a single band on an electrophoretic disc-gel. The molecular weight of the enzyme was estimated to be 44, 000 Da by SDS-polyacrylamide gel electrophoresis, and 45, 000 Da by gel filtration, suggesting the monomeric state of the enzyme. The isoelectric point of the enzyme was about pH 4.5. The optimal pH and temperature for the enzyme reaction were 6.0 and $50^{\circ}C$, respectively. The purified enzyme digested inulin into di-D-fructofuranose-l, 2': 2, 3'-dianhydride, confirming the enzyme was an inulin fructotransferase (inulinase II).

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.121-126
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• 1996

A bacterial strain LC-413, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose dianhydride(DFAIII) and amount of oilgosaccharides, was isolated from soil and pre-sumed as Flavobacteium sp. LC-413. The enzyme production was induced by inulin as carbon source and enhanced by the addition of 0.3% malt extract and 0.2% {TEX}$NaNO_{3}${/TEX} as nitrogen source. The enzyme activity in the culture supernatant reached at the maximum, 78.6units/ml, after 11 hours of cultivation in the medium composition of 1.5% inulin, 0.2% {TEX}$NaNO_{3}${/TEX}, 0.05% {TEX}$K_{2}${/TEX}{TEX}$HPO_{4}${/TEX}, 0.05% {TEX}$MgSO_{4}${/TEX}.7{TEX}$H_{2}${/TEX}O, 0.05% KCI, a trace amount of {TEX}$FeSO_{4}${/TEX}.7{TEX}$H_{2}${/TEX}O, and 0.3% malt ext. at 3$0^{\circ}C$. The oilgosaccharide produced by enzyme reaction from inulin was identified as DFA III by and {TEX}${13}^C${/TEX}-NMR spectrosocpy.

• Applied Biological Chemistry
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• 제40권3호
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• pp.184-188
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• 1997

Inulin을 가수분해하여 di-D-fructofuranose dianhydride(DFA)를 생성하는 inulin frucroransferase를 분비하는 균주를 토양으로부터 분리하였으며, Bacillus sp로 동정하였다. 본 효소에 의하여 생성되는 DFA는 TLC 와 HPLC로 분석한 결과, fructose 두 분자가 ${\beta}\;1,2':{\alpha}2,3'$ 결합을 한 DFA III로 동정되었다. 본 균주는 탄소원으로 1.5% 돼지감자즙, 유기 질소원으로 1.0% peptone, 무기 질소원으로 $0.27%\;NH_4H_2PO_4$를 사용했을 떼 효소 생산이 2.709 units/ml로 최대였으며, inulin을 탄소원으로 사용할 경우는 1.0% yeast extract, $0.2%\;NaNO_3$를 첨가했을 때 효소 생산이 2.245units/ml로 최대를 나타내었다. 본 균주를 최적 액체 배지에서 72시간 배양한 결과 배양 45시간에 2.61 units/ml 최대 활성을 보였다.

• Journal of Microbiology and Biotechnology
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• 제10권2호
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• pp.275-280
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• 2000

The inulin fructotransferse (depolymerizing) (IFTase, EC 2.4.1.93) gene of Arthrobacter sp. A-6 was cloned and expressed in Escherichia coli and Bacillus subtilis. The IFTase gene consisted of an ORF of 1.311 nucleotides encoding a polypeptide of 436 amino acids containing a signal peptide of 31 amino acids in the N-terminus. The molecular mass of the IFTase based on the nucleotide sequence was calculated to be 46.116 Da. The recombinant E. coli $DH5{\alpha}$ cells expressing the Arthrobacter sp. A-6 IFTase gene produced most of the IFTase intracelularly. In contrast, the recombinant B. subtilis DB 104 carrying the IFTas gene on a B. subtilis-E. Coli expression vector secreted the IFTase into the culture fluid efficiently.

• Applied Biological Chemistry
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• 제36권2호
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• pp.105-110
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• 1993

Enterobacter sp. S45로부터 inulin fructotransferase를 생산, 정제하고 효소적 특성을 조사하였다. 배양상징액의 $0.4{\sim}0.8$ 포화$(NH_4)_2SO_4$ 침전물인 조효소는 DEAE-cellulose column chromatography 및 fast protein liquid chromatography로 정제하였으며 효소의 회수율은 0.9%였고 약 148배의 정제도를 보였다. 정제된 효소는 전기영동상으로 단일 band였으며 분자량은 SDS-PAGE에 의해 42,800으로 나타났다. 이 효소의 최적 pH는 5.5, 최적 온도는 $50^{\circ}C$였으며 $Mg^{2+}$이온은 효소활성을 30% 증가시키나 $Hg^{2+}$, $Cu^{2+}$ 및 $Fe^{3+}$이온들은 활성을 강력히 저해하였다. Inulin에 대한 km 값은 1.4 mM, Vmax 값은 $0.196\;{\mu}mole/min$이었다. 본 효소는 inulin을 fructose 말단으로부터 fructose 두 분자씩 절단, 환상형인 DFA를 생성하며, 그 결과 inulin 분해산물인 GF, $GF_2$, $GF_3$, $GF_4$ 등도 검출되었다. 중합도에 따른 효소활성을 조사해 본 결과 이 효소는 중합도 $4(GF_3)$ 이상의 fructo 올리고당에만 작용하여 $GF_3$는 DFA III와 GF로, $GF_4$는 DFA III와 $GF_2$로 변환시켰다. 또한 본 효소는 sucrose, raffinose 및 melezitose를 기질로서 이용하지 못하므로 invertase 및 ${\alpha}-glucosidase$ 활성이 없는 것으로 나타났다.

• 한국미생물·생명공학회지
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• 제27권6호
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• pp.471-476
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• 1999

Inulin fructotransferase(depolymerizing)(EC 2.4.1.93)(inulaseII) which converts inulin into di-D-fructofuranose-1,2':2,3'-dianhydride (DFAIII) was purified from Arthrobacter ureafaciens KCTC 3387 using column chromatography on DEAE-Toyopearl 650M and gel filtration of Sephadex G-200. The enzyme was purified 7-fold with a yield of 11% from a culture supernatant. The purified enzyme gave a single band on polyacrylamide gel electrophoresis, and the molecular weight of the enzyme was estimated to be 45,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the enzyme reaction were pH6.5~7.0 and $55{\circ}C$, respectively. The enzyme was stable within a pH range of 5.0 to 10.6 and up to $60^{\circ}C$. The Km of this enzyme for DFAIII production was 11.9mM. The enzyme was inactivated by $Hg^{2+}$ and after exhaustive digestion of inulin by this enzyme, 1-kestose and nystose were produced in addition of DFAIII.

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.619-623
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• 1996

Some physical and physiological properties of di-D-fructofuranose dianhydrideIII (DFAIII), as a new sweetener, were investigated via in vitro experiments. The disaccharide was prepared by decomposing inulin with inulin fructotransferase (depolymerizing) from Arthrobacter sp. A-6. DFAIII had more excellent heat and acid stability than sucrose. This was one of the most desirable properties especially for the oligomer types of sweetener. DFAIII showed the least pH drop in the Streptococcus mutans culture, compared with the other saccharides examined. This indicates that the sugar will be fairly effective for preventing dental caries. The saccharide also had a selective Bifidus growth-promoting effect in PYF medium. Whereas, E. coli did not show growth promotion in the DFAIII-containing medium. In the co-culture of Bifidus longum and E. coli in the BL medium, Bifidus longum had a selective growth while the growth of E. coli appeared rather to be inhibited.