• KSBB Journal
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• 제14권5호
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• pp.539-542
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• 1999

의 대량생산과 분리.정제 기술의 개발을 위해 Arthrobacter ureafaciens KCTC 3387 균주를 이용한 발효 및 분리정제된 효소반응을 통한 DFAIII의 생산과 회수에 대하여 조사하였다. 첫 번째 방법으로는 Arthrobacter ureafaciens 균주를 발효배양하여 DFAIII 양이 최고가 되는 시점에서 발효정지 시킨후 silica gel를 통해 gel filtration을 분리하였고, 두 번째 방법으로는 정제된 효소로 반응시킨 다음 silica gel을 통해 gel filtration을 하여 분리하였으며, 세 번째 방법으로는 발효배양상등액에 에탄올을 첨가하여 생산물을 침전시켜 DFAIII를 분리하였다. 25 g/L의 초기 inulin 농도로부터 각각 1.57, 4.40, 0.34 g/L 의 정제분말이 얻어져 각각 6.3, 17.6, 1.4%의 수율로 회수되었고, 81, 97, 87%의 순도를 각각 나타내었다. 효소반응을 통한 DFAIII 생산 및 회수가 가장 높은 수율과 순도로 회수되었으며, 발효배양을 통한 DFAIII의 생산은 초기기질농도 25 g/L의 50%에 해당하는 DFAIII가 생산되었으나 효소반응에 의한 생산보다는 낮은 수율로 회수되었고 순도는 세 가지 방법 중 가장 낮았으며, 에탄올 침전반응은 가장 낮은 수율을 나타내었다.

• 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.

• 한국미생물·생명공학회지
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• 제25권3호
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• pp.277-285
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• 1997

Kluyveromyces marxianus var. marxianus isolated as an inulin-assimilating microorganism produces inulin fructotransferase (inulaseII) which catalyses the conversion of inulin into di-D-fructofuranose 1, 2' : 2, 3' dianhydrde (DFAIII). The DFA produced by the organism was isolated by using active carbon column, and identified as DFAIII by high performance liguid chromatography. The culture medium giving maximum inulaseII production was found to consist of 1% sucrose and 0.75% yeast nitrogen base (YNB). The inulasell production was induced by inulin or sucrose as a carbon source and increased by addition of YNB as a nitrogen source. Optimal initial pH of the culture medium, culture temperature and medium volume for the enzyme production were pH 4.7, 30$\circ$C and 140 ml, respectively. Under the optimal conditions described above, the enzyme activity in the culture supematant reached 4.2 units/ml after cultivation for 36 h. The DFAIII was accumulated at 13.25 mg/ml after 48 h of culture in the Jerusalem artichoke tuber medium.

• 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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• 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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• 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.

• 미생물학회지
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• 제36권1호
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• pp.69-73
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• 2000

Chicory 뿌리에서 Arthrobacter sp. A-6를 배양하여 inulin fructotransferase를 생산하였을 매 균주의 성장속도, 효소의 생산성을 검토하였다. 가공된 치커리 뿌리는 inulin fructotransferase의 crude enzyme solution으로 처리하였을 때 생산되는 di-fructofuranose dianhydride(DFA III)의 양을 chicory 뿌리의 가공방법에 따른 차이를 비교하였다. 우선 standard 배지에서 생산된 효소액으로 10%의 inulin이 포함된 standard inulin용액을 처리하면, 1.14mg/ml의 DFA III가 생산되었다. Chicory뿌리의 전처리방법에 따라, 같은 조건으로 반응을 진행시키는 실험을 통해 각 배지에서의 생산효율을 비교하였다. Chicory뿌리를 washing과 extraction과정을 거치지 않고 그대로 반응시켰을 경우, 2.29 mg/ml의 DFA III가 생산되어 생산성이 가장 높았는데, 이는 세척과정에서 inulin이 유실되지 않으므로, 기질로 작용하는 inulin의 양이 가장 높은 데 기인하는 것으로 생각된다.