• Journal of Microbiology and Biotechnology
• /
• v.16 no.3
• /
• pp.360-367
• /
• 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
• /
• v.33 no.4
• /
• pp.281-287
• /
• 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.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.33 no.2
• /
• pp.305-310
• /
• 2004

The present study was aimed at investigating effects of chicory inulin and three kinds of oligosaccharides on lipid metabolism in rats fed a high-cholesterol diet. Nine Sprague-Dawley male rats weighing, about 190g were given one of five experimental diets, which were basal cholesterol diet (Control) isomaltooligosaccharide diet (IMO), Iructooligosaccharide diet (FO), chicory inulooligosaccharide diet (CIO) and chicory inulin diet (CI) for 5 weeks. In the oligosaccharide and inulin diets, 6% was added at the expense of sucrose. Rats were pair-fed to the intake of FO group which consumed the least amount, and their feces were collected during the last 4 days. Body weight gain was lower in Fo and CI groups compared with the Control group. Plasma glucose levels of FO and CIO groups were lower and plasma triglyceride concentrations of FO, CIO, and CI groups were lower than those of IMO group. Plasma cholesterol concentration did not differ among groups. Relative liver weight was lower in CIO group. Hepatic triglyceride and cholesterol did not differ among. groups. Fecal excretion of neutral steroid and bile acid were not different among groups, but fecal triglyceride excretion was significantly increased in FO and CI groups compared with the Control group. In conclusion, supplementation of oligosaccharides and chicory inulin at 6% of diets showed no significant hypolipidemic effect in rats fed a high cholesterol diet.