Abstract
A cellulolytic enzyme (pI 4.3) was isolated from culture filtrates of Trichoderma reesei QM 9414 by column chromatography and preparative flat bed isoelectric focusing. The focusing experiments were carried out using an extremely shallow pH gradient (pH 3.7–4.6), which made it possible to recover one single protein band out of numerous Trichoderma isoenzymes and/or multi-enzyme complexes in the acidic pH region. The purified hydrolase is homogeneous as assessed by analytical isoelectric focusing, SDS gel electrophoresis, and titration curve analysis under detergent/ urea conditions. It was found to have an apparent molar mass of 68 000 g mol −1 (SDS-PAGE) and showed its optimum activity towards cellotetraose at pH 4 and 60°C. It is a glycoprotein with a carbohydrate content of 27% (as glucose). The hydrolase does not act with an endo-mechanism (CMC as substrate) and splits off mainly cellobiose from microcrystalline cellulose. In the current literature the enzyme would thus be termed an exo-cellobiohydrolase [1,4-β-D-glucan cellobiohydrolase (CBH), EC 3.2.1.91]. However, when the enzyme's action pattern was further evaluated using defined soluble cellodextrins (DP 2–7) as substrates, it became obvious from the recorded concentration-time course data that the enzyme does not exclusively split off cellobiose units from the oligomers. In conclusion, we do not believe that a cellobiohydrolase analogous to β-amylase (EC 3.2.1.2) exists, at least in the cellulase system of Trichoderma reesei. The term CBH or cellobiohydrolase should thus not be used any longer in the literature.
Published Version
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