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6 CH 2 H H H CH 2 n-1 H CH 2 n

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8 H Potato α-glucan phosphorylase H n soluble starch P i n-1 H P() 2 glucose-1-phosphate Pi H CBPase H glucose P i glucose-1-phospate cellobiose H cellooligosaccharide n+1 CDPase n

9 H N 2 CDPase H N 2 p-nitrophenyl-β-d-glucoside G-1-P P i PNP-Cellooligosaccharide n Reaction conditions p-nitrophenyl-β-d-glucoside 30 mm Glucose-1-phosphate 80 mm DTT 10 mm EDTA 1 mm CDPase 1 U/ml 25 mm Tris-HCl buffer (ph 9.0) Temperature 50ÞC Time 12 hr sup. ppt Soluble PNP-cellooligosaccharides Insoluble PNP-cellooligosaccharides

10 NH H H α α

11 α α β β α α H CH 2 NH H

12 β CH 2 NH H β n CH 2 H NH β n Condition for Synthesis of Cellulase Inhibitors. 20 mm G1(β-1,4)DNJ 80 mm Glucose-1-phosphate 50 mm Tris-HCl (ph 7.5) 1 mm EDTA 10 mm DTT 40 units/ml CDP Temperature 50ÞC Reaction volume 5.0 ml Yield of Purified Inhibitors Synthesized by the Reverse Reaction of CDP Phosphorolysis Yield Inhibitor (mg) (mol%) G2(β-1,4)DNJ G3(β-1,4)DNJ G4(β-1,4)DNJ G5(β-1,4)DNJ G6(β-1,4)DNJ Total 49.2 G1(β-1,4)DNJ G2(β-1,4)DNJ G3(β-1,4)DNJ G4(β-1,4)DNJ G5(β-1,4)DNJ G6(β-1,4)DNJ Reaction time (hr) Thin-layer Chromatogram of Cellulase Inhibitors Synthesized by the Reverse Reaction of CDP.

13 Inhibition Cellulase G1-DNJ G2-DNJ G3DNJ G4-DNJ G5-DNJ G6-DNJ Endo-type cellulases Aspergillus aculeatus FI-CMCase Trichoderma viride cellulase II-b Irpex lacteus endo-type cellulase (En-1) Cryptococcus flavus CMCase Bacillus sp. N-4 cellulase (NK1) Bacillus subtilis cellulase (BSC) Cellulomonas uda CB4 CMCase Exo-type cellulases Aspergillus aculeatus cellobiohydrolase I Aspergillus aculeatus avicelase III + + Trichoderma viride cellulase III Irpex lacteus exo-type cellulase (Ex-1) The endo-type cellulase (0.25 ml; 0.08 units/ml) was incubated with 0.25 ml of each inhibitor ( M) with 0.5 ml of 1.0%CMC for 10 min. The exo-type cellulase (0.05 ml; 0.06 units/ml) was incubated with 0.05 ml of each inhibitor ( M) with insoluble celloorigosaccharides for 60 min. Inhibitory ratings used were as follows: +++, %; ++, 50-74%; +, 25-50%;, 0-24%.

14 Inhibitors K i (M) Aspergillus aculeatus β-1,4-glucanase 1 G2(β-1,4)DNJ G3(β-1,4)DNJ G4(β-1,4)DNJ G5(β-1,4)DNJ G6(β-1,4)DNJ Trichoderma viride β-1,3-glucanase G1(β-1,3)DNJ Aspergillus sp. endo-β-1.6-glucanase G1(β-1,6)DNJ

15 Hydrolysis products of the inhibitors Cellulase G1-DNJ G2-DNJ G3DNJ G4-DNJ G5-DNJ G6-DNJ Endo-type cellulases A. aculeatus FI-CMCase T. viride cellulase II-b G2 G2>>G1 G2>>G1 G2>>G1 G2>>G1 D G1D G1D G1D G1D I. lacteus endo-type cellulase (En-1) G3>G2 G2=G3 G2=G3 G1D>G2D G1D=G2D G1D=G2D C. flavus CMCase B. sp. N-4 cellulase (NK1) G3>G2 G2=G3 G2=G3 G1D>G2D G1D=G2D G1D=G2D B. subtilis cellulase (BSC) G2=G3 G3>G2 G2=G3 G2=G3 G1D>G2D G1D>G2D G2D>G1D G1D=G2D C. uda CB4 CMCase Exo-type cellulases A. aculeatus cellobiohydrolase I G2 G2 G2 G2 G2 D G1D D G1D D A. aculeatus avicelase III G2 G2>G3 G2>G3 G2>G3 G1D G2D>G1D G2D>G1D G2D>G1D T. viride cellulase III G2 G2>G3 G2 G2D G3D>G2D G2D I. lacteus exo-type cellulase (Ex-1) G2>G1 G2>G3 G2=G3 G2=G3 G1D>G2D G2D>G1D G2D>G1D G2D>G1D 40 μl of each of the inhibitors ( M) and 40 μl of the enzyme (endo-type, 0.16 units/ml; exo-type, 0.12 units/ml) were incubated for 12 h, and then the reaction was stopped by heating for 30 min in boiling water. The products was analyzed by TLC., not detected; G1, glucose; G2, cellobiose; G3, cellotriose; D, 1-deoxynojirimycin (DNJ); G1D, G1( β-1,4)dnj; G2D, G2(β-1,4) DNJ; G3D, G3(β-1,4)DNJ.

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18 H H βα CH 2 H βα H CH 2 H βα βα

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(Shigen to Sozai) Vol.116 p (2000) 石炭灰フライアッシュからのゼオライトのアルカリ水熱合成と生成物の陽イオン交換特性 * 1 1 村山憲弘山川洋亮 2 3 小川和男芝田隼次 Alkali Hydrothermal Synthesis of Zeol 石炭灰フライアッシュからのゼオライトのアルカリ水 Title 熱合成と生成物の陽イオン交換特性 Author(s) 村山, 憲弘, 山川, 洋亮, 小川, 和男, 芝田, 隼次 Citation 資源と素材 : 資源 素材学会誌, 116(4): 279-284 Issue Date 2000 URL http://hdl.handle.net/10112/5454 資源 素材学会 (http://www.jstage.jst.go.

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