Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates

FEBS Letters

Claeyssens, Marc¹  van Tilbeurgh, Herman¹ 

[1] Laboratorium voor Biochemie, Faculteit Wetenschappen, State University of Ghent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

关键词: Cellulase;    Exo-cellobiohydrolase;    Endocellulase;    Assay;    Trichoderma reesei;    Fluorogenic substrate;    Isoelectric focusing;    PAG-IEF;    polyacrylamide gel isoelectric focusing;    endocellulase;    1;    4-β-glucan glucanhydrolase (EC 3.2.1.4);    exo-cellobiohydrolase (CBH);    1;    4-β-glucan cellobiohydrolase (EC 3.2.1.91);    MeUmbβGlc;    4-methylumbelliferyl β-D-glucopyranoside;    MeUmbβ-(Glc) n (n = 2;    3);    4-methylumbelliferyl β-D-glycosides of cellobiose and cellotriose;    MeUmb β-Gal β(1→4) Glc;    4-methylumbelliferyl β-lactoside;    MeUmb;    4-methyl-umbelliferone;    HPLC;    high-pressure liquid chromatography;   

DOI  :  10.1016/0014-5793(85)81260-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The 4-methylumbelliferyl β-D-glycosides of glucose, cellobiose, cellotriose and lactose are used to differentiate several exo-cellobiohydrolase, endocellulase and β-glucosidase activities in crude cellulase from Trichoderma reesei. Spectrophotometric or fluorimetric assays allow simple detection and quantitative measurements of eluate activities from ion-exchange chromatography and after analytical gel electrofocusing. Using the fluorophoric glucoside several β-glucosidases can be visualised after isoelectric focusing on polyacrylamide gels. The use of the lactoside and of the same substrate supplemented with cellobiose as inhibitor allows a clearcut distinction to be made between endocellulase II and exo-cellobiohydrolase I. Both enzymes are present as ‘iso-enzyme’ mixtures. With the cellotrioside only one fraction is detectable (endocellulase III). The same methods could be used in culture growth experiments.

【 授权许可】

Unknown