Papain digestion of crude Trichoderma reesei cellulase: purification and properties of cellobiohydrolase I and II core proteins.
The major cellulase components produced by Trichoderma reesei are composed of distinct catalytic and cellulose-binding domains. A simple two-step procedure is described for the purification of the catalytic domains, also termed core proteins (cp), of the major components, cellobiohydrolase (CBH) I and II. The novel aspect of this procedure is that native CBH I and II do not have to be purified initially. Papain digestion of a commercial T. reesei cellulase preparation followed by gel filtration on a Superdex 75 column resulted in the separation of fractions containing CBH I cp and CBH II cp; chromatofocusing purified the latter to homogeneity. N-terminal protein sequencing of CBH II cp provided good evidence for its identity. A comparison of the catalytic activity and cellulose-binding ability of these cp was made. A major difference between them was that CBH II cp bound to microcrystalline cellulose, unlike CBH I cp. CBH I cp readily hydrolysed the bond between the aglycone and cellobiose in p-nitrophenyl cellobioside unlike the CBH II cp preparation. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyse barley beta-glucan. It was also shown that incubation of cellulose fibres with native CBH I, CBH I cp or CBH II cp resulted in a smoothing of the fibre surface.
Woodward
J
J
Chemical Technology Division, Oak Ridge National Laboratory, TN 37831-6194.
Brown
J P
JP
Evans
B R
BR
Affholter
K A
KA
D000327:Adsorption; D000595:Amino Acid Sequence; D002384:Catalysis; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology*
D002482:Cellulose; D006026:Glycoside Hydrolases; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D010206:Papain