• Title, Summary, Keyword: levanase

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Isolation and Cultivation of Microorganism Producing Levanheptaose from Levan (Levan으로부터 Levanheptaose를 생산하는 미생물의 분리 및 배양)

  • Lim, Young Soon;kang, Soo Kyung;Kang, Eun Jung;Lee, Tae Ho
    • Korean Journal of Microbiology
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    • v.34 no.1_2
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    • pp.37-42
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    • 1998
  • The microorganisms degrading levan were screened from soil. The isolated strain produced levanase releasing single oligosacchride from levan. The optimum cultural medium for levanase production (g/l) was composed of 0.5% levan, 0.1% $K_2HPO_4$, 0.05% NaCl, 0.3% $NaNO_3$, 0.3% yeast extract (pH 8.0). The cultivation for levanase production was carried out in 500 ml shaking flask containing 50 ml of the optimum medium at $30^{\circ}C$ on a reciprocal shaker, and the highest levanase production was observed after 54 hours of cultivation. The levanase hydrolyzed levan into single oligosaccharide. The product purified by chilled EtOH precipitation and gel filtration was detected as a single peak on HPLC analysis. The oligosaccharides formed by enzyme reaction was identified as levanheptaose (DP7) by HPLC and by ESI-MASS.

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Isolation and Cultivation of Microorganism Producing Levanbiose from Levan (Levan으로부터 Levanbiose를 생산하는 미생물의 분리 및 배양)

  • 이태호;강은정;강수경
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.27 no.3
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    • pp.441-447
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    • 1998
  • A bacterial strain No. 43 was isolated from soil samples as a levan-assimiating microorganism producing an extracellular levanase and hydrolying levan to levanbiose. According to the taxonomic characteristics of its morphological and physiological properties, the strain was idenified as Pseudomonas sp. No. 43. The optimum culura medium was composed of 10g levan, 5g(NH4)2SO4, 3g NH4Cl, 3g polypepton, 1g K2HPO4, 0.5gMgSO4.7H2O, and 0.2g MnCl2.4H2O per liter. The cultivation for levanase was carried out in pH 7.0 at 4$0^{\circ}C$ for 28hr. The reaction product was a kind of oligosaccharide and it was purified by chilled ethanol precipitation and gel filtration for evalluation of degree of polymerization (DP). The purified product was determined as levanbiose of MW 342 and DP2 by HPLC and FAB-MS.

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Molecular Cloning and Nucleotide Sequence of Endo-Inulinase Gene from Xanthomonas oryzae #5

  • Kim, Byeong-U;Kim, Mi-Rang;Yu, Dong-Ju
    • 한국생물공학회:학술대회논문집
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    • pp.655-659
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    • 2000
  • A 11.5-kb DNA fragment containing an endo-inulinase gene was cloned from Xanthomonas oryzae #5. It contained a single open reading frame of 3,999bp, encoding a polypeptide composed of signal peptide of 32 amino acids and mature protein of 1,301 amino acids. From the comparison of amino acids sequences with fructan hydrolases, inulinase, levanase and CFTase, the sequence of the endo-inulinase had highly homology of 72% with CFTase of B. circulans, and six highly conserved regions including the ${\beta}-fructosidase$ motif were found.

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Growth Effect of Levanoligosaccharide(Levanoctaose) on Intestinal Microflora (Levanoligosaccharide(levanoctaose)의 장내미생물에 대한 생육효과)

  • 강수경;박나희;이태호
    • Korean Journal of Microbiology
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    • v.35 no.2
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    • pp.153-157
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    • 1999
  • The effececl of levanoclaose produced by levanase Croni Pseuc/olol~~o!!n.s sp. K-52 on pnnciple inlesimal microflorawas investigated. The reaction product, levanoctaose, was used as a carbon source for various intestinalmicroflora. Especially. Bijidobacteriulolr~ adolescentis and Lnctohaciilrrs ocidophilus grew effectively in vitroexpeiiments, whereas Clostridiunz per.frilol~gerzs, Bactetvid,~ngilis, Eschericlzin. coli, and Stnplzylococcus nureirsdid not. Therefore, levanoctdose seemed to proinole selectively the growth ol" B. ndo1escenti.r and L. acidol~hi-/us. In Lhe in viva experiments, the effects of levanoctaose on inlestinal nucroflora were examined on heirgrowth. $\beta$-fri~ctosidase acliviiy. and butyrate concenuation in rats. Appuently, die number of fecal Bifidobacteria.the amount of bulyrate, and $\beta$-hctosidase activity were increased, whereas total aerobes 'and pH werereduced in rals Eed leviu~octaose diets, cornpxed with hose of the control diets. We concluded hat those effecismay be beneficial in improving gastrointestinal health.astrointestinal health.

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Physiological Effects of Levanoligosaccharide on Growth of Intestinal Microflora (Levanoligosaccharide의 장내미생물의 생육에 미치는 생리효과)

  • 이태호;강수경;박수제;이재동
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.29 no.1
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    • pp.35-40
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    • 2000
  • The effect of levanheptaose produced by levanase from Streptomyces sp. 366L on principle intestinal microflora was investigated. The reaction product, levanheptaose, was used as a carbon source for various intestinal microflora. As a results, Bifidobacterium adolescentis, Lactobacillus acidophilus, and Eubacterium limosum grew effectively in the in vitro experiment, whereas Clostridium perfringens, E. coli, and Staphylococcus aureus did not. Therefore levanheptaose seems to promote selectively the growth of B. adolescentis and L. acidophilus. In the in vivo experiment, the effect of levanheptaose on the growth of intestinal microflora, $\beta$-fructosidase activity, pH, and butyrate concentration were examined in rats. Apparently, the number of fecal Bifidobacteria, the amount of butyrate, and $\beta$-fructosidase activity were increased, whereas total aerobes and pH were reduced in rats fed by levanheptaose diets, compared with those of control diets. We concluded that those effects may be beneficial in improving gastrointestinal health.

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Probing the Critical Residues for Intramolecular Fructosyl Transfer Reaction of a Levan Fructotransferase

  • Moon, Keum-Ok;Choi, Kyoung-Hwa;Kang, Ho-Young;Oh, Jeong-Il;Jang, Se-Bok;Park, Cheon-Seok;Lee, Jong-Hoon;Cha, Jae-Ho
    • 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.

Identification of catalytic acidic residues of levan fructotransferase from Microbacterium sp. AL-210 (Microbacterium sp. AL-210이 생산하는 levan fructotransferase의 효소활성에 중요한 아미노산의 동정)

  • Sung, Hee-Kyung;Moon, Keum-Ok;Choi, Ki-Won;Choi, Kyung-Hwa;Hwang, Kyung-Ju;Kim, Myo-Jung;Cha, Jae-Ho
    • Journal of Life Science
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    • v.17 no.1
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    • pp.6-11
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    • 2007
  • [ $\beta$ ]-Fructofuranosidases, a family 32 of glycoside hydrolases (GH32), share three conserved domains including the W(L/M)(C/N)DP(Q/N), FRDPK, and ECP(D/G) motifs. The functional role of the conserved acidic residues within three domains of levan fructotransferase, one of the $\beta-fructofuranosidases$, from Microbacterium sp. AL-210 was studied by site-directed mutagenesis. Each mutant was overexpressed in E. coli BL21(DE3) and purified by using Hi-Trap chelating affinity chromatography and fast performance liquid chromatography. Substitution of Asp-63 by Ala, Asp-195 by Asn, and Glu-245 by Ala and Asp decreased the enzyme activity by approximately 100-fold compared to the wild-type enzyme. This result indicates that three acidic residues Asp-63, Asp-195, and Glu-245 play a major role in catalysis. Since the three acidic residues are present in a conserved position in inulinase, levanase, levanfructotransferase, and invertase, they are likely to have a common functional role as nucleophile, transition state stabilizer, and general acid in $\beta-fructofuranosidases$.