• KSBB Journal
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• v.14 no.5
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• pp.539-542
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• 1999

For the mass production of DFAIII and for the development of techniques of separation and purification of it, the methods of production of DFAIII and its recovery was investigated by fermentation with the strain of Arthrobacter ureafaciens KCTC 3387 and by enzyme reaction. In the first method, DFAIII was produced by fermentation with the strain of Arthrobacter ureafaciens KCTC 3387 and recovered from culture supernatant with silica gel gy filtration, in the second method, it was produced by enzyme reaction and recoverd with the same method of the first, and in third method it was produced by fermentation and recovered by addition of ethanol to the culture supernatnat.Against 25g/L of initial concentration of inulin, 1.57, 4.40, 0.34 g/L of powder of DFAIII was recovered respectively and the rate of recovery was 6.3, 17.6 1.4% and the purity was estimated at 81, 97, 87% respectively. For the production of DFAIII and its recovery, enzyme reaction method was the highest in the rate of recovery and its purity. By fermentation method, DFAIII was produced with 50% fo initial concentration of substrate but th rate of recovery was lower than enzyme reaction method and purity was lowest among the three methods. Ethanol pricipitation method showed the lowest rate of recovery.

• Microbiology and Biotechnology Letters
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• v.24 no.5
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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.

• Microbiology and Biotechnology Letters
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• v.25 no.3
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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.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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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.

• Microbiology and Biotechnology Letters
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• v.27 no.6
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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.

• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.36-40
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• 1993

A bacterial strain, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose dianhydride (DFA) was isolated from soil and presumed as Enterobacter sp. The DFA isolated on Bio-gel P2 column was identified as DFA III by high performance liquid chromatography and $^13C-nmr$ spectroscopy. The enzyme production was induced by inulin and markedly enhanced by the addition of corn steep liquor and $NH_4H_2P0_4$ for nitrogen source. Under optimum condition, the enzyme activity in the culture broth reached at maximum, 0.22 unit/ml after cultivation for 72 hour.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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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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• v.1 no.1
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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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• v.40 no.3
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• pp.184-188
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• 1997

A bacterial strain, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose 1,2':2,3' dianhydride(DFA III), was isolated from soil and presumed as Bacillus sp.. The highest production of the enzyme was obtained by using medium containing Jerusalem artichoke extract as carbon source, peptone as organic nitrogen source, and $NH_4H_2PO_4$, as inorganic source. Under optimum condition, the enzyme activity of the culture broth supernatant reached maximal 2.61 units/ml after cultivation for 45 hrs.

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.69-73
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• 2000

Arthrobacter sp. A-6 was cultivated and DFA III(di-fructofuranose dianhydride) was produced with inulin fructotransferase from the chicory root. The specific growth rate, yield of cell mass and yield of enzyme from the culture in variable chicory root extracts were studied and the results compared. Standard inulin solution(10%) was treated with the crude enzyme solution of inulin fructotransferase from the cell culture, 1.14mg/ml of DFA III was produced. The enzyme reactions were processed with various preparations of chicory root extracts in the same conditions. The highest yield of DFA III production(2.29 mg/ml) was obtained from the chicory roots without washing or extraction. The yield of DFA III from the washed chicory roots without extraction was at lowest(0.44 mg/ml). The production process of inulin fructotransferase and DFA III from the chicory root without prewashing or extraction steps were more efficient.