• Journal of Microbiology and Biotechnology
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• v.6 no.6
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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.

• 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.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.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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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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• v.36 no.2
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• pp.105-110
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• 1993

Inulin fructotransferase from Enterobacter sp. S45 was purified with DEAE-cellulose column chromatography and fast protein liquid chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was estimated to be 42,800 by SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature for the enzyme reaction were pH 5.5 and $55^{\circ}C$, respectively. $Mg^{2+}$ activated the enzyme activity, but $Fe^{3+}$, $Cu^{2+}$, $Hg^{2+}$ significantly inhibited. After exhaustive digestion of inulin by the enzyme, DFA III, sucrose, 1-kestose and nystose were produced. Sucrose, 1-kestose, raffinose and melezitose can't be used as substrates by the enzyme, but nystose and 1-F-fructofuranosyl nystose were hydrolysed. The Km and Vmax for inulin of the enzyme were 1.4 mM and $0.196\;{\mu}mole/min$, respectively.

• 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.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.