| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1996: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
1.Simultaneous saccharification and fermentation of Jerusalem artichoke tubers were conducted batchwise at 30ﾟC using Aspergillus niger 817 and Saccharomyces cerevisiae 1200. Ethanol concentrations obtained were 10.4% (v/v) from the ground tubers after 15 h, 15.0% from the juice concentrate after 72 h, and 20.1% from the flour after 120 h.
2.In a screening of inulinolytic fungi, Penicillium sp.TN-88 was selected as the best producer of inulinase. The extracellular endoinulinase P-II was purified to be homogeneous, as judged by SDS-polycrylamide gel electrophoresis, with an apparent M_r of 68.0 kDa. The specific activity was 105 U/mg. The enxyme activity was highest at pH 5.2 and 50ﾟC.The enzyme hydrolyzed inulin to the extent of 70% and liberated inulotriose as the main product, but lacked activity toward sucrose, raffinose or levan. The apparent K_m value for inulin was 0.20 mM at 40ﾟC and pH 5.0 The N-terminal amino acid sequence of the enzyme was 1-DDYRPAFHFC PAENXMNEPN GLIQIXSTXH-30.
3.Two genomic genes encoding endoinulinase from Aspergillus niger 12 were cloned in E.coli and sequenced. Southern-blot analysis indicated that the endoinulinase genes, inuA and inuB,were present on separate EcoRI-digested fragments of sizes 3.9 kbp and 5.9 kbp, respectively. Each fragment contained a single open reading frame of 1,548 bp, and no intervening sequences were found within the coding region. The mature enzymes consisted of 493 amino acids and was preceded by a putative signal peptide of 23 amino acids. The enzymes contained two Cys residues and six potential sites for N-linked glycosylation. The cell-free extracts of E.coli JM109 harboring the cloned inuA and inuB genes showed inulinase activities of 0.42 and 0.59 U/mg of protein, respectively. The deduced amino acid sequences of the mature A.niger enzymes showed 73% identity with that of the Penicillium purpurogenum endoinulinase.
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