• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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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

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.281-287
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• 2005

Paenibacillus polymyxa 유래의 endoinulinase 유전자(inu, 2.733 kb, E.C 3.2.1.7)를 Saccharomyces cerevisiae에 발현시키기 위해 대장균과 효모의 shuttle vector (GALl promoter함유)에 subcloning하여 구축된 pYGENIU27 (8.6 kb) plasmid를 S. cerevisiae SEY2102에 형질전환하였다. Uracil이 결핍된 SD배지와 inulin이 포함된 배지에서 효모 형질전환체를 선별하였다. 효모 형질전환체의 배양에서 endoinulinase는 periplasmic space에 다량 존재함이 확인되었다. YPDG배지에서 재조합 endoinulinase는 총 활성 1.81unit/ml으로 생산되었다. Inulooligosaccharides (IOSs) 생산을 위한 효소의 최적 반응 조건으로 pH 8.0,반응온도 $45^{\circ}C$, 기질은 Jerusalem artichoke로 결정되었다. 효소활성은 pH 10.0에서도 안정적으로 유지되었으며, 최적 반응 조건 (ginulin당 36 unit효소 이용)에서 반응 10분 후부터 IOSs가 생산되기 시작하였다. 생성된 IOSs의 구성분으로 inulobiose (F2), inulotriose (F3)와 inulotetraose (F4)가 생성되었고, 이중에서 F3가 주성분이었다. 이상의 결과는 효모에서 생산된 재조합 endoinulinase를 이용하여 inulin으로부터 기능성 감미소재인 IOSs의 산업적 생산을 위한 기초결과로 활용될 것이다.

• Journal of Microbiology and Biotechnology
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• 제8권3호
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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 Applied Biological Chemistry
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• 제42권2호
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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.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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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.