• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1064-1069
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• 2008

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose-2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.64-67
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• 2006

To enhance the stability of ascorbic acid, the glycosylation of ascorbic acid was studied using the transfructosylation activity of levan fructotransferase. When levan was used as glycosyl donor, a novel fructoside (ascorbic acid 2-ffuctoside) was formed by the transfructosylation activity of the levan fructotransferase. The production of ascorbic acid 2-fructoside was highly affected by the concentration of the fructosyl acceptor (ascorbic acid). When $35\%$ of ascorbic acid and $2\%$ of levan were incubated with LFTase of 0.5 unit/glevan at $37^{\circ}C$ for 85 h, a maximum 52 g/l of AA-2F was produced.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.99-104
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• 2005

Microbacterium laevaniformans levansucrase synthesized various hetero-oligosaccharides by transferring fructosyl residue from sucrose to various saccharides as acceptors. The acceptor specificity test showed that reducing saccharides were more favorable acceptors than nonreducing saccharides. The transfructosylated product, fructosyl galactose, was produced in the presence of D-galactose as an acceptor. The chemical structure of the resulting fructosyl galactose was analyzed by yeast invertase and NMR, and identified as O-$\alpha$-D-galactosyl-(1${\to}$2)-$\beta$-D-fructofuranoside. These results indicate that the main transfructosylation activity of the enzyme is to make nonreducing transferred products via a transfer of fructosyl residue to acceptor molecules having reducing group. When nonreducing sugars, such as methyl $\alpha$-D-glucoside and methyl $\alpha$-D-galactoside, were used as an acceptor, the transfer product was also formed in spite of the reducing group blocked with methyl group. The fact that no transfer product was formed with sugar alcohols as acceptors was suggested to be due to marked conformational difference of acceptors.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.251-257
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• 1998

Cycloinulooligosaccharide fructanotransferase (CFTase) which produces cyclofructan from inulin was purified 332-fold from a culture broth of Bacillus macerans CFCl. The molecular mass of the CFTase was estimated to be 110 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration, indicating that the enzyme has a monomer structure. The maximal level of enzyme activity was observed at pH 7.5 and $45^{\circ}C$. The enzyme was stable in the pH range 6.0 to 9.5, and at temperatures up to $45^{\circ}C$ for 1 h. The enzyme activity was completely inhibited in the presence of 0.5 mM $Ag^+\;or\;Cu^2+$ ion. None of sucrose (GF), l-kestose (GF2), or nystose (GF3) were found to be substrates for the CFTase, but inulooligosaccharides larger than nystose were attacked by the enzyme. The CFTase catalyzes not only the cyclization as the major reaction, but also disproportionation and coupling reactions involving intermolecular transfructosylation in the same manner as cyclodextrin glucanotransferase (CGTase) (EC 2.4.1.19).

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.877-880
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• 2000

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides consisting of six to eight molecules $\beta$-($2\rightarrow1$)-linked cyclic D-fructofuranose through intramolecular transfructosylation. We have examined the regulation of CFTase synthesis in Bacillus macerans and Bacillus subtilis. Synthesis of the CFTase was induced by inulin and it was subject to carbon catabolite repression (CCR) by glucose in both microorganisms. The DNA sequence upstream of the promoter of the CFTase gene was not involved in the inulin induction and glucose repression of the CFTase gene expression in B. subtilis. This suggests that the DNA element(s) responsible for the inuline induction and glucose repression is located downstream of the promoter region. Unexpectedly, the CCR of the expression of CFTase gene was observed not to be dependent on CcpA protein in B. subtilis.

• Food Science and Biotechnology
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• v.16 no.3
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• pp.482-484
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• 2007

To investigate production and hydrolysis of levan, the levansucrase enzymes from Zymomonas mobilis ATCC 10988 and Rahnella aquatilis ATCC 33071 were used. The optimum temperature of R. aquatilis levansucrase for levan formation was $37^{\circ}C$, whereas that of Z. mobilis was $4^{\circ}C$, under the experimental conditions. Both levansucrases also catalyzed the reverse levan hydrolysis reaction. Levan hydrolysis reactions from both levansucrases were temperature dependent; high temperature ($20^{\circ}C$) was more favorable than low temperature ($4^{\circ}C$) by 4 times. Fructose was the only product from hydrolysis reaction by both levansucrases, showing that both levansucrases mediated the hydrolysis reaction of exo-enzyme acting. In both enzymes, initial levan hydrolysis activity was almost accounted to 1% of initial levan formation activity. The results allow the estimation of the fructose release rate in enzyme processing conditions.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.309-315
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• 1998

An extracellular levansucrase, which catalyzes the formation of levan from sucrose, from the culture broth of Zymomonas mobilis ZM1 was purified by conventional column purification methods. The final purification yield was 18.3 fold of the crude enzyme from Z. mobilis, with 16.5 % of the enzyme recovered in the preparation step. The molecular weight of the enzyme was estimated to be 91,000 by Superose 12 gel filtration, and 45,000 by SDS-PAGE, indicating that levansucrase is a dimer. The optimum pH for the enzyme activity was around pH 4.0 for sucrose hydrolysis, and was around pH 5.0 for levan formation. The enzyme was inhibited by some metal ions, such as Hg$\^$2+/ and Cu2$\^$2+/, and 50% of inhibition was observed with 5mM EDTA. The enzyme activity was enhanced by the presence of detergent Triton X-100, but inhibited by SDS completely The enzyme catalyzes the liberation of reducing sugars, oligosacccharides and the formation of fructose polymer(levan). The enzyme also catalyzes the transfructosylation reaction of fructose moiety from sucrose to various sugar acceptor molecules, including sugar alcohols.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.921-928
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• 2002

Microorganism producing extracellular CFTase was isolated from soil and designated as Bacillus polymyxa MGL21. The gene encoding the CFTase (cft) from B. polymyxa MGL21 was cloned and sequenced. The ORF of the cf gene was composed of 3,999 nucleotides, encoding a protein (1,333 amino acids) with a predicted molecular mass of 149,375 Da. Sequence analysis indicated that CFTase was divided into five distinct regions. CFTase contained three regions of repeat sequences at the N-terminus and C-terminus. The endo-inulinase region of homology (ERH) of CFTase was similar to that of Pseudomonas mucidolens endo-inulinase ($50\%$ identity, 259 amino acids). Furthermore, CFTase possessed a highly conserved core region, which is considered to be functional for the hydrolysis reaction of inulin. The cft gene was expressed in a His-tagged form in Escherichia coli cells, and the His-tagged CFTase was purified to homogeneity. The optimal temperature and pH for CFTase activity were found to be $50^{\circ}C$ and 9.0, respectively. The enzyme activity was completely inhibited by 10 mM $Ag^+\;and\;Cu^2+$. Thin-layer chromatography analyses indicated that CFTase catalyzed not only the cyclization reaction ut also disproportionation and hydrolysis reactions as well.