A cellulose-binding module of the Trichoderma reesei beta-mannanase Man5A increases the mannan-hydrolysis of complex substrates.
Endo-beta-1,4-D-mannanases (beta-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The beta-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5ADeltaCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5ADeltaCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.
Hägglund
Per
P
Department of Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden.
Eriksson
Torny
T
Collén
Anna
A
Nerinckx
Wim
W
Claeyssens
Marc
M
Stålbrand
Henrik
H
D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D002482:Cellulose / Q000737:chemistry*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D006868:Hydrolysis; D008351:Mannans / Q000737:chemistry*; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D014242:Trichoderma / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D044902:beta-Mannosidase
D002352:Carrier Proteins; D008351:Mannans; D011134:Polysaccharides; D021122:Protein Subunits; D011994:Recombinant Proteins; C007916:hemicellulose; D002482:Cellulose; D008361:Mannosidases; D044902:beta-Mannosidase