CH 2 H H H CH 2 n-1 H CH 2 n
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
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
NH H H α α
α α β β α α H CH 2 NH H
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 10 10.3 G3(β-1,4)DNJ 9 7.2 G4(β-1,4)DNJ 11 5.6 G5(β-1,4)DNJ 18 9.3 G6(β-1,4)DNJ 38 16.8 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 0 2 4 6 8 Reaction time (hr) Thin-layer Chromatogram of Cellulase Inhibitors Synthesized by the Reverse Reaction of CDP.
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 (1.0 10-4 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 (1.0 10-3 M) with insoluble celloorigosaccharides for 60 min. Inhibitory ratings used were as follows: +++, 75-100%; ++, 50-74%; +, 25-50%;, 0-24%.
Inhibitors K i (M) Aspergillus aculeatus β-1,4-glucanase 1 G2(β-1,4)DNJ 6.9 10-6 G3(β-1,4)DNJ 1.9 10-7 G4(β-1,4)DNJ 3.8 10-8 G5(β-1,4)DNJ 2.8 10-7 G6(β-1,4)DNJ 3.3 10-7 Trichoderma viride β-1,3-glucanase G1(β-1,3)DNJ 3.8 10-5 Aspergillus sp. endo-β-1.6-glucanase G1(β-1,6)DNJ 9.3 10-5
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 (1.0 10-3 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.
α
H H βα CH 2 H βα H CH 2 H βα βα
H H βα
2010-020746
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