• KSBB Journal
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• v.21 no.2
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• pp.140-143
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• 2006

Acid- and enzymatic hydrolysis properties of two fructans(inulin and levan) and their oligofructoses has been investigated. At pH 1, the initial fructose release rate differs and is rapidly hydrolyzed in the order of levan oligosaccharide and inulin oligosaccharide, levan, inulin. At pH 4.5, 7 and 14, no or little amount of fructose are found from four samples. At the presence of inulinase in the reaction mixture, the fructose is rapidly produced from all samples, whilst invertase treatments show low activities. The results allow the estimation of the fructose release rate in many foodstuff processing conditions.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.528-530
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• 2002

We have previously reported recombinant deletion mutant was constructed from cycloinulo-oligosaccharide fructanotransferase(CFTase) gene of Xanthmonas oryzae MGL21. Purification of the mutant protein from E. coli and reation condition for the production of inulo-oligosaccharide(ISO) were studied. The molecular mass of the CFTase deletion mutant protein was estimated to be 90kDa by SDS- Polyacrylamide gel electrophoresis. Optimum reaction pH and temperature were pH6.5 and $40^{\circ}C$, respectively. Under optimal reaction conditions, endo-inulinase activating enzyme was rapidly produced ISO from inulin. Components were DP(degree of polymerization) 3and 4 with trace amount of smaller oligosaccharides.

• Journal of Applied Biological Chemistry
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• v.42 no.2
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• pp.71-74
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• 1999

The crude enzyme prepared from the culture supernantant of Arthrobacter sp. S37 was purified by Phenyl Toyopearl column chromatography. Six endo-inulinases were detected by activity staining on native PAGE and named Inu I to Inu VI. Endo-inulinase were further purified by DEAE cellulose column chromatography and band slicing. Inu II~VI produced mainly inulotriose (F3) and inulotetraose (F4) as well as a small amount of inulobiose (F2) and fructose in contrast to Inu I producing F3, F4 and F5 from inulin. The N-terminal amino acid sequence of native and six CNBr-cleaved fragment of Inu VI were determined. No homology was found in amino acid sequences between Inu VI and other fructan hydrolase including invertase reported.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.253-258
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• 1991

- An exoinulinase (EC 3.2.1.80) was purified from a commercial inulinase preparation from Aspergillus ficuum using ion exchange chromatography on CM-Sephadex C-50 and DEAESepharose 6B and HPLC gel filtration on a Protein Pak 125 column. Native exoinulinase had a molecular weight of 83, 000$\pm$ 1, 000 and was glycoprotein. Optimal pHs of the enzyme were ranged from 4.4 to 4.7. About ninety five percent of the whole activity was maintained even after incubation of 8 hours at $55^{\circ}C$.The enzyme was a typical non-specific P-fructofuranosidase, of which I/S ratio appears to be 0.35.

• KSBB Journal
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• v.8 no.1
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• pp.10-16
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• 1993

The use of Jerusalem artichoke containing $\beta$-1, 2-fructose oligomer for the production of D-sorbitol and L-sorbose has been studied. The employment of inulinase(0.398%, v/v) for the hydrolysis of 40% (v/w) Jerusalem artichoke juice resulted in 36.7g/1 of glucose and 85.3g/1 of fructose at $50^{\circ}C$. These sugars were utilized as substrates for D-sorbitol and L-sorbose production. Coimmobilization of inulinase and permeabilized cells of Zymomonas mobilis in the mixture of chitin (5%, w/e) and x-carrageenan(4%, w/v) resulted in the production of 30.2g/1 of D-sorbitol by using inulin as a substrate. The process of L-sorbose production from D-sorbitol by Gluconobacter suboxydans was optimized with respect to the substrate concentration, level of dissolved oxygen and glucosic and concentration. Gluconlc acid produced by Zymomonas mobilis from glucose was found to inhibit Gluconobacter suboxtans in conversion of D-sorbitol to L-sorbose. In view of removing such inhibitory effect by gluconic acid, mutants were selected by the NTG (N-methyl-N'-N'-nitro-N-nitrosoguanidlne) treated method. Mutants selected by NTG mutagenesis showed no inhibitory effects of gluconic acrid against L-sorbone production when its concentration increased up to 100g/1. A mutant produced 40.1g/l of L-sorbose in the medium containing 100g/l D-sorbitol and 100g/l-gluconic acid. This result is consider able when compared with L-sorbose concentration (21.7g/1) obtained from the fermentation with wild type strain of Gluconobacter suboxnians.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.19 no.5
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• pp.519-526
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• 2018

Fructan, a fructose homopolymer, is found in various foods, including onion, garlic, chicory, Jerusalem artichoke, banana, and Cheonggukjang. This study aimed to quantitatively analyze both levan-type and insulin-type fructan using acid analysis and enzyme treatment. In order to analyze fructan contents, we applied optimized conditions to various fructan-rich foods using products from 2017. In the case of oxalic acid hydrolysis, fructose concentrations increased as oxalic acid concentration increased. Inulinase treatment was better than invertase treatment in terms of fructose liberation from fructan. We applied three different methods to fructan-rich foods, including onion, garlic, banana, and Cheonggukjang and found that fructose released from fructan-rich foods was the highest in oxalic acid hydrolysis among three different methods. Except for Cheonggukjang, inulinase treatment produced better results in terms of fructose contents than invertase treatment. From our study, estimated daily fructan intakes by Koreans were 1,172~3,402 mg from onion and garlic. In conclusion, we believe that information on fructan-rich foods may be helpful to understand their roles in the human digestive system.

• 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.

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

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.4
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• pp.242-246
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• 2000

Human lipocortin-I was expressed as a secretory product by Saccharomyces cerevisiae harboring an expression system consisting of GAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromato-graphy. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys26.

• Journal of Microbiology and Biotechnology
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• v.15 no.1
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• pp.202-205
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• 2005

Our previous study showed that the overexpression of carboxypeptidase Y (CPY) of Saccharomyces cerevisiae in Escherichia coli resulted in the formation of insoluble inclusion bodies. To produce soluble CPY, we designed a novel Pichia pastoris expression system, in which the following were inserted into expression vectors: three different signal sequences derived from the mating factor a1 of S. cerevisiae, an inulinase of Kluyveromyces marxianus, and the endogenous signal sequence of CPY. The expression vector pHIL-D2-SSinul-proCPY was the most effective in the production of proCPY among the vectors examined. The purified active CPY was obtained from proCPY by treating with proteinase K, followed by QExcellose ion-exchange column chromatography.