• Microbiology and Biotechnology Letters
• /
• v.19 no.1
• /
• pp.52-56
• /
• 1991

Inulin degradation was examined using patially puriiied enzyme mixtures of the Exo-inulinase from a Bacillus spp. and the Endo-inulinase from a Pseudomonas spp.. The highest synergistic xtion of the two cnzymcs was observcd when the Exo- and the Endo-inulinase werc mixed at the ratio of 1 to 13, and the rate of hydrolysis of the above process was enhanced approximately 1.6 times I1ight.1- than that of the reaction catalysed with a single enzyme of the same units. The enzymc mixture showed the maximal activity at pH 6.0 and $55^{\circ}C$, and in the prescncc of 0.5 mM each of $CO^{2+}$ and $Mn^{2+}$. Under the optimal condition described above fructosu was accumulated with the overitll concentration of 84% after 36 hours of the reiiction.

• Journal of Life Science
• /
• v.9 no.1
• /
• pp.22-28
• /
• 1999

A bacterium producing exo-inulinase was isolated from soil and identified Pseudomonas sp. and named as Pseudomonas sp. NO5. The optimal culture conditions for the efficient production of exo-inulinase from Pseudomonas sp. NO5 were obtained by cultivating with the medium 1$\%$ sucrose, 0.5$\%$ yeast extract, 0.5$\%$ $(NH_4)_2$$HPO_4$, 0.05$\%$ $MgSO_4$ㆍ$7H_2$0, 0.001$\%$ and $FeSO_4$ㆍ$7H_2$0 at $37^{\circ}C$ in initial pH 7.0 for 20 hours. The enzyme was induced maximally in the presence of sucrose or inulin at early stationary phase about 20 hour after cultivation.

• Microbiology and Biotechnology Letters
• /
• v.18 no.5
• /
• pp.490-495
• /
• 1990

The extracellular inulinase from Bacillus spp. was purified to a single protein through a sequence of operations including ammonium sulfate fractionation, heat treatment, DEAE Sepharose C1-6B ion exchange chromatography, Sephadex 6-100 and Sephadex 6-150 gel filtration. The purified enzyme was confirmed to be a $\beta$ -D-fructofuranosidase(EC 3.2.1.26) which was much more active on sucrose than on inulin(I/S = 0.2). The maximal inulinase activity was observed at pH 6.0 and at the temperature of $50^{\circ}C$. The mo1ecular weight of the enzyme was about 56, 000. Tryptophan and histidine residues of the enzyme molecule were found to be essential for its catalytic activity.

• Proceedings of the Korean Society of Life Science Conference
• /
• 2002.03a
• /
• pp.90-90
• /
• 2002

• Microbiology and Biotechnology Letters
• /
• v.20 no.1
• /
• pp.20-24
• /
• 1992

In order to reuse inulinase effectively, a method for immobilizing both endo- and exoinulinase to vinylsulfone activated agarose via covalent bond was investigated. The immobilized enzyme preparation had, respectively, 400 U for exoinulinase activity and 80 U for endoinu- Iinase activity per gram gel. A thermal stability by immobilization had increased in the case of exoinulinase. Optimum pHs for two immobilized enzymes were 4.4 to 5.0. Synergistic effect which depends on mixed ratio of two immobilized enzymes was the best when the mixed ratio of endo/exo lay between 0.1 and 0.5, and its activity of the mixed enzyme increased 1.7 times as compared to that of each immobilized enzyme. Inulinase activities of both of the immobilized enzymes did not change during 20 times experimental runs in a batch reactor.

• Journal of Life Science
• /
• v.8 no.5
• /
• pp.614-621
• /
• 1998

An extracellular inulinase from Penicillium sp. which isolated from soil sample was purified to a single protein th-rough ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography and Toyopearl HW 65 F gel filtration. The temperature and pH for the enzyme reaction were around 6$0^{\circ}C$ and 4.0, respectively. The enzyme was stable at 3$0^{\circ}C$-5$0^{\circ}C$ and in the pH range of 4 to 5. $CuCl_2$, $HgCl_2$ and EDTA inhibited the enzyme activity strongly. By contrast, $MnCl_2$ and $CaCl_2$ activated the enzyme activity. The molecular weight of the purified enzyme was esti-mated to be 77,000 dalton by SDS-polyacrylamide gel electrophoresis. The Km values of the enzyme for inulin were calculated to be $2.2\times10^{-3}$M. TLC analysis suggested that purified enzyme is exo-type inulinase. The NH2-terminal amino acid sequences of the purified enzyme was determined to be $NH_2$-X-Glu-Ser-Tyr-Thr-Glu-Lys/Leu-Tyr-Arg-Pro.

• Journal of Life Science
• /
• v.19 no.9
• /
• pp.1218-1225
• /
• 2009

Difractose anhydrides (DFAs) is studied as a sweetener for diabetics because of its structural property. DFAs have four types: DFA I, III, IV (degradation of levan) and V (degradation of inulin). Especially, DFA IV has been shown to enhance the absorption of calcium in experiments using rats. Levan fructotransferase is an enzyme for producing di-d-fructose-2,6':6,2-dianhydride (DFA IV). To identify structural characterization, we purified wild-type and mutants (D63A, D195N and N85S) of levan fructotransferase (LFTase) from Microbacterium sp. AL-210. These proteins were purified to apparent homogeneity by Ni-NTA affinity column, Q-sepharose ion exchange and gel filtration chromatography and detected by SDS-PAGE. They were also analyzed by circular dichroism (CD) measurements, JNET secondary structure prediction, activity measurements at various temperatures, and pH analysis. The optimum pH for the enzyme-catalyzed reaction was pH 7.5 and optimum temperature was observed at $55^{\circ}C$. Along with wild-type LFTase, mutants were analyzed by CD measurement, fluorescence analysis and differential scanning calorimetry (DSC). N85S showed less $\alpha$-helix and more $\beta$ strand than others. Also, N85S showed almost the same curve as wild-type in their steady-state fluorescence spectra, whereas mutant D63A and D195N showed higher intensity than wild-type. The amino acid sequence of wild-type LFTase was compared to the sequences of exo-inulinase from Aspergillus awamori, a plant fructan 1-exohydrolase from Cichorium intybus, and Thermotogo maritime (Tm) invertase and showed a high identity with Exo-inulinase from Aspergillus awamori.