• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.18-22
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• 1993

An assay system by using antibody was adopted to screen the endoinulinase producingfungi due to its high specificity toward endoinulinase, To determine whether the affinity-purified rabbit serum, which were generated against the purified endoinulinase, can react only with the endoinulinase, rocket immunoelectrophoresis was performed. The results showed that the serum specifically reacts with endoinulinase but not with exoinulinase and other proteins in the culture media. Using this polyclonal antibody, a strain from 62 fungal colonies was selected and it secreted an endoinulinase in the culture media to the amount comparable to that of Aspergillus ficuum A Tee 16882 known as a high endoinulinase producer.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.587-589
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• 2000

The production of inulooligosaccharides from inulin by a dual endoinulinase system of Pseudomonas sp and Xanthomonas sp. was investigated the optimum conditions for a dual endoinulinase reaction were as follows : pH,5.8; temperature, $50^{\circ}C$; substrate concentration, 50 g/l; enzyme ratio, 3:1 as Xanthomonas endoinulinase to Pseudomonas endoinulinase. Under optimum conditions, the maximum yield of oligosaccharides was 90.5% in total sugar basis by dual endoinulinase system

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.281-287
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• 2005

The endoinulinase gene (inu, 2.733 kb, EC 3.2.1.7) from Paenibacillus polymyxa was subcloned into an Escherichia coli-yeast shuttle vector with GALl promoter for the expression in Saccharomyces cerevisiae. The constructed plasmid, pYGENIU27 (8.6 kb) was introduced into S. cerevisiae SEY2102 cell and then the yeast transformant was selected on the synthetic defined media lacking uracil and on the inulin-containing media. The recombinant endoinulinase was predominantly localized in the periplasmic space of the yeast cell. The total activity of the endoinulinase reached 1.81 unit/ml by cultivation of yeast transformant on YPDG medium. The optimized conditions determined for the inulooligosaccharides (IOSs) production from inulin were as follows; pH, 8.0; reaction temperature, $45^{\circ}C$; inulin source, Jerusalem artichoke. Enzyme activity was stably maintained up to the pH of 10.0. Under the optimized condition and with endoinulinase of 36 unit/g-inulin, IOSs started to be produced after 10 min of enzymatic reaction. By the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), and inulotetraose (F4) were produced and F3 was the major product. Consequently, these data would be used as a fundamental parameters for the production of functional sweetener IOSs from inulin by recombinant yeast endoinulinase.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.478-481
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• 2000

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase free of an exoinulinase and an extracellular invertase in the culture medium. When inulin was included in the medium, a recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony. An expression of endoinulinase was dependent on the culture temperature and shaking. The highest expression of endoinulinase was observed at $30^{\circ}C$, and 150rpm.

• KSBB Journal
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• v.13 no.3
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• pp.284-288
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• 1998

Production of inulo-oligosaccharides from inulin was carried out with the maximum yield of 94% using a purified endoinulinase from Aspergillus ficcum. The optimum reaction temperature and pH were 55-60$^{\circ}C$ and pH 5.5-6.0, respectively. The Michaelis constant and maximum reaction velocity of the endoinuinase for inulin were 13.27 g/L and 0.13 g/L$.$min at 55$^{\circ}C$, pH 5.5, respectively. Inulin source had no significant effect on oligosaccharide yield and product composition, although initial production rate differed according to inulin origins. The reaction pH was a critical factor in inulo-oligosaccharide production because considerable free monosaccharides were released, decreasing oligosaccharide yield at low pH conditions. An empirical relationship describing the reaction performance was developed from kinetic data: the time to reach maximum oligosaccharide yield (tw) as a function of initial concentration of inulin (lo) and enzyme (Eo); i.e., log tM = -1.025 log Eo - 0.011 l0 + 2.655.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.360-367
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• 2006

The endoinulinase gene (inu1) from Pseudomonas mucidolens was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The inu1 gene of P. mucidolens was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTENIU (8.5kb), was then introduced to S. cerevisiae EBY100 cells and the yeast transformants selected on synthetic defined media lacking uracil and inulin-containing media. The inu1 gene under the control of the GAL1 promoter was successfully expressed in the yeast transformants, and the surface display of endoinulinase confirmed by immunofluorescence microscopy, along with its enzymatic ability to form inulooligosaccharides (IOSs) from inulin. The total endoinulinase activity reached about 2.31 units/ml when the yeast transform ants were cultivated on a YPDG medium. To efficiently hydrolyze the inulin, various reaction conditions were examined, including the pH, temperature, and inulin source. The optimized conditions were then determined as follows: pH, 7.0; temperature, $50^{\circ}C$; inulin source, Jerusalem artichoke. Under the optimized condition and 46 units of endoinulinase per g of inulin, IOSs started to be produced after 10 min of enzymatic reaction. The highest yield, 71.2% of IOSs, was achieved after 30 h of reaction without any significant loss of the initial enzyme activity. As a result of the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) were produced, and F4 was the major product.

• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.158-162
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• 1991

Endoinulinase was purified from a commercial inulin preparation produced by Aspergillus ficuum using ion exchange chromatography on CM-Sephadex C-50 and DEAE-Sepharose 6B, HPLC gel filtration on a Protein Pak 125 Colum and HPLC ion exchange chromatography on a TSK DEAE-5pw Column. The endoinulinase had a molecular weight of 72,000${\pm}$1,000 and was glycoprotein with 23 to 25% w/w sugar content. The enzyme was much more active on inulin with random cleavage mode than on sucrose and on palatinose: The ration of activity on inulin and sucrose (I/S ratio) was 10~14.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.385-385
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• 2005

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

• 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.