Studies of the cellulolytic system of Trichoderma reesei QM 9414

Abstract

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II,58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel).By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH I1 core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from ~r~c~o d e r m a reesei [Teeri et al. (1987) Gene 51,43 -521. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH TI in terms of adsorption and catalytic properties, Distinct functions can be attributed to the terminal peptides: for intact CBH I1 the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH I1 synergism experiments.The filamentous fungus Trichoderma reesei presents an ideal system for the study of cellulose hydrolysis since it produces a variety of enzymes including endocellulase and exocellulase (cellobiohydrolases) and b-glucosidases (cellobiases) [l]. The primary structure of several of these proteins is presently known [2 -61 but no structure/function relationships have yet been established. The two cellobiohydrolases, CBH I and CBH I1 [7 -111, lack any apparent homology in their amino acid sequence except for a conserved region present at their C and N termini respectively [4].Their action is similar both with regard to their strong adsorption onto insoluble substrates and lack of carboxymethylcellulase activity. However, studies both with microcrystalline cellulose [9] and small, soluble substrates [I 21 reveal some differences in specificity. In addition, the two cellobiohydrolases interact synergistically both with each other and with endoglucanases on microcrystalline substrates In order to differentiate these enzymes further by their function and to investigate possible domain structure, proteolysis studies were performed. Preliminary results on the limited action of papain on native CBH I have been reported by us [16]. Two fragments were isolated: first, a heavily glycosylated peptide, tentatively identified with the C terminus; secondly, a core protein containing the active (hydrolytic) site. The terminal peptide was characterised as an independent structural and functional dom...

Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw

“…The specific activities of the six major enzymes obtained, the two cellobiohydrolases CBH1 (Cel7a) and CBH2 (Cel6a), the major two endoglucanases EG1 (Cel7b) and EG2 (Cel5a), as well as the xyloglucanase Cel74a and the xylanase XYN1, were measured on Avicel, carboxymethylcellulose (CMC), xylan and xyloglucan (Table 1). The two cellobiohydrolases have high activity on Avicel cellulose, consistent with values found in the literature [20,21] whereas endoglucanases and XYN1 show lower activities. A low activity of xylanase on Avicel and CMC has been reported previously [22].…”

A functionally based model for hydrolysis of cellulose by fungal cellulase

“…The rate constants for CBH1 and CBH2 (k CBH1 and k CBH2 ) were used based on the best fit to the data from the experimental data. The specific activity of CBH2 was twice that of CBH1 (Tomme et al, 1988). The rate constant for EG1, k EG1 , was set at fivefold that used for CBH1 in light of the relative specific activity of these two enzymes, mechanistic considerations, and experimental data fitting (Tomme et al, 1988;Zhang and Lynd, 2004).…”

“…The specific activity of CBH2 was twice that of CBH1 (Tomme et al, 1988). The rate constant for EG1, k EG1 , was set at fivefold that used for CBH1 in light of the relative specific activity of these two enzymes, mechanistic considerations, and experimental data fitting (Tomme et al, 1988;Zhang and Lynd, 2004). The typical composition of unfractionated T. reesei cellulase mixtures was taken to be as reported elsewhere (Goyal et al, 1991) such that 100 mg cellulase contains 60 mg CBH1, 20 mg CBH2, and 12 mg EG1.…”

The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

“…It has also been shown that deletion of the CBH 1 gene has a drastic effect on the cellulolysis of T. reesei [3]. Most fungal cellulases have a common structural organization where the main part of the enzyme, the catalytic core, is connected through a heavily glycosylated linker region to a small cellulose-binding domain (CBD) [4]. This gives the whole molecule an elongated tadpole shape (180 A in the case of CBH 1) [5].…”