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Papain digestion of crude Trichoderma reesei cellulase: purification and properties of cellobiohydrolase I and II core proteins
Author(s) -
Woodward J.,
JP Brown,
BR Evans,
KA Affholter
Publication year - 1994
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1994.tb00291.x
Subject(s) - cellulase , trichoderma reesei , cellobiose , chemistry , cellulose , hydrolysis , papain , chromatography , sephadex , size exclusion chromatography , chromatofocusing , beta glucosidase , biochemistry , enzyme
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.