The purification and properties of the C1 component of Trichoderma koningii cellulase

Abstract

1. The C(1) component that was isolated from a Trichoderma koningii cellulase preparation (Wood, 1968) by chromatography on DEAE-Sephadex with a salt gradient was still associated with a trace of CM-cellulase activity (determined by reducing-sugar and viscometric methods). 2. Further chromatography on DEAE-Sephadex, with a pH gradient instead of a salt gradient, provided a C(1) component that could still produce reducing sugars from a solution of CM-cellulose (to a very limited extent), but which could no longer decrease the viscosity (i.e. under the assay conditions employed). 3. No evidence for the non-identity of C(1) component and the trace of CM-cellulase activity could be found when electrofocusing was done in a stabilized pH gradient covering three pH units (pH3-6) or, alternatively, only 0.5 pH unit (pH3.72-4.25). 4. The two protein peaks that were separated by electrofocusing in carrier ampholytes covering only 0.5 pH unit (isoelectric pH values of 3.80 and 3.95) were shown to be isoenzymes of the C(1) component: they differed in the extent to which they were associated with carbohydrate (9% and 33%). 5. The purified C(1) component had little ability to attack CM-cellulose or highly ordered forms of cellulose, but degraded phosphoric acid-swollen cellulose readily: cellobiose was the principal product of the hydrolysis (97%). 6. Dewaxed cotton fibre was degraded to the extent of 15% when exposed to high concentrations of C(1) component over a prolonged period: cellobiose was again the principal sugar present in the supernatant (96%). 7. Cellotetraose and cellohexaose were hydrolysed almost exclusively to cellobiose. 8. Evidence indicates that the C(1) component is a beta-1,4-glucan cellobiosylhydrolase.

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems

“…Bacterial cellulose (BC) has an average DP of 2,000 -3,000 (Hestrin, 1963;Fierobe et al, 2002;Valjamae et al, 1999), while bacterial microcrystalline cellulose (BMCC) prepared by treatment of BC with acids ranges from 130 -1,300, depending on hydrolysis conditions (Valjamae et al, 1999). The DP of phosphoric-acid swollen cellulose (PASC) ranges from 30 to more than 1,000 Krassig, 1985;Petre et al, 1981;Wood and McCrae, 1972), depending on the DP of the starting substrate (Wood, 1988;Hoshino et al, 1997), as well as the phosphoric acid incubation time and temperature (Krassig, 1993).…”

Oxidation: An important enzyme reaction in fungal degradation of cellulose

“…It was later suggested that the Cr -enzyme was an exe-1,4-&glucanase [ 14,151. This is supported by Wood and McCrae [ 161 and Berghem and Pettersson [ 171. It is now understood that the en&-l ,4-P-glucanases act at random over the cellulose chain and the exo-1,4+enzyme acts on exposed chain ends splitting off cellobiose [16,17] …”

Bacterial genomes: what they teach us about cellulose degradation

“…In the era before DNA sequencing and cloning, this method was the sole route to obtaining and characterizing novel cellulases [10][11][12][13][14][15][16][17][18][19]. In this early work, protein sequence data was either not reported or a limited N-terminal sequence was published; making matching of purified enzymes to gene sequences extremely challenging.…”