Peter Tomme

Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei: Separation of functional domains

Limited proteolysis of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei by papain yields a core protein (56 kDa) which is fully active against small, soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. Activity against an insoluble substrate, such as Avicel, is however completely lost and concomitantly decreased adsorption onto this microcrystalline cellulose is observed. The peptide (10 kDa), initially split off during proteolysis, is identified as the heavily glycosylated carboxy‐terminal of the native CBH I. Depending on the experimental conditions the core protein is further nicked in between disulfide bonds, but its properties and stability do not appreciably differ from those of intact CBH I. These results lead to the proposal of a bifunctional organisation of the CBH I: one domain, corresponding to the carboxyterminal, acts as a binding site for insoluble cellulose and the other, localised in the core protein, contains the active (hydrolytic) site.

Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei.

Monoclonal antibodies have been produced against two functionally different domains present in two cellobiohydrolases from Trichoderma reesei (CBH I and CBH II). Four groups of antibodies were obtained, which specifically recognized (Western blotting, ELISA) (a) the core protein within CBH I, (b) the core protein within CBH II, (c) the BA region of CBH I, and (d) the ABB region of CBH II. No cross-reactivities within these four groups were observed. The antibodies reacted also specifically with proteins of similar size to CBH I and CBH II (SDS-PAGE) from other Trichoderma strains (Western blotting), whereas no reaction was observed with cellulases from other fungal sources. Analysis of culture filtrates of T. reesei QM 9414 harvested at various times of growth on cellulose under buffered conditions (pH 5-6) indicated the presence of only single bands of CBH I and CBH II, even after prolonged cultivation (160 h). Cultivation on cellulose in unbuffered media, however, showed the appearance (Western blotting) of additional lower molecular weight proteins, which reacted with the monoclonal antibodies directed against the cores of CBH I and II, but not with those recognizing the respective BA and ABB regions. The appearance of these lower molecular weight bands was most pronounced in unbuffered media, supplemented with a 3-fold (w/w) amount of organic nitrogen (peptone). Analysis of some commercial cellulase preparations from T. harzianum revealed the same pattern of lower molecular weight proteins, in contrast to samples from other fungal cellulases. Those samples or preparations, showing a multiple pattern of CBH I and CBH II, exhibited higher activities of an acid proteinase. These results imply that the use of unbuffered, high nitrogen-supplemented culture conditions for production of cellulases may lead to considerable proteolytic modification of the secreted cellobiohydrolases.

Domain structure of cellobiohydrolase II as studied by small angle X-ray scattering: Close resemblance to cellobiohydrolase I

Evidence for a domain structure of cellobiohydrolase II (CBH II, 58 kDa) from Trichoderma reesei (Teeri et al., 1987; Tomme et al., 1988) is corroborated by results from SAXS experiments. They indicate a tadpole structure for the intact CBH II in solution (Dmax = 21.5 +/- 0.5 nm; Rg = 5.4 +/- 0.1 nm) and a more isotropic, ellipsoid shape for the core protein (Dmax = 6.0 +/- 0.3 nm; Rg = 2.1 +/- 0.1 nm). The latter was obtained by partial proteolysis with papain which cleaves the native CBH II to give two fragments (Tomme et al., 1988): the core (45 kDa) with the active (hydrolytic) domain and a smaller fragment (11 kDa) coinciding with the tail part of the model and containing the binding domain for unsoluble cellulose. This peptide fragment is conserved in most cellulolytic enzymes from Trichoderma reesei (Teeri et al., 1987). It contains a conserved region (block A) and glycosylated parts (blocks B and B duplicated and located N-terminally in CBH II). In spite of different domain arrangements in CBH I (blocks B-A at C-terminals) SAXS measurements (Abuja et al., 1988) indicate similar tertiary structures for both cellobiohydrolases although discrete differences in the tail parts exist.

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl β‐D‐cellotrioside (K m = 48μM, k cat = 0.7 min−1) with release of the β‐cellobiose (retention of configuration). The same enzyme catalyzes the (trans‐hydration of cellobial (K m = 116 μM, K cat = 1.16 min−1) and lactal (K m = 135 μM, k cat = 1.35 min−1), presumably with glycosyi oxo‐carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl β‐cellotrioside, but products formed from these prochiral substrates are again of β configuration. Cellobiohydrolase II from the same microrganism hydrolyzes methyl β‐D‐cellotetraoside (K m = 4 μM, k cat = 112 min−1) with inversion of configuration to produce α‐cellobiose. The other reaction product, methyl β‐cellobioside, is in turn partly hydrolysed by Cellobiohydrolase II to form methyl β‐D‐glucoside and D‐glucose, presumably the α‐anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si‐direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei A chemical modification study

Several aspects of the specific modification of cellobiohydrolase I (CBH I) from Trichoderma reesei with Woodwards reagent K (N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate) are presented. The pH dependence of the resulting inactivation points to the implication of an ionising group with a pK a of approx. 5.5. The rapid inactivation kinetics, the specific protection and the stoichiometry of modification (3 versus 2 residues), together with the isolation and amino acid sequencing of the putative active site peptide, provide a large body of evidence for the presence of a catalytically important carboxyl residue in the 125–135 region of the CBH I amino acid sequence. From the striking homology between this peptide sequence and those of the active site regions of different lysozymes, glutamic acid 126 is retained as the most plausible catalytic residue (proton donor) in CBH I, equivalent to glutamic acid 35 in hen egg white lysozyme. Glutamic acid 127 is proposed as a potential active site residue to the homologous endoglucanase I (EG I) isolated from the same Trichoderma species.

Chromatographic separation of cellulolytic enzymes

Publisher Summary Numerous fractionation problems have been encountered with cellulolytic complexes from microorganisms. Ion-exchange chromatography and isoelectric focusing (chromatofocusing) are the methods used most frequently. Using the parent cellobioside ligand a simple method for the purification of endo- and exocellulases from different sources was obtained. In the case of the enzymes from Trichoderma reesei and Penicillium pinophilum specific elution allows group separation of functionally related enzymes that are known to appear as isoenzymes in the culture filtrates. The relative affinity for the ligand p -aminobenzyl-l-thioceliobioside is shown to determine the applicability of the method in the isolation of an endocellulase from Clostridium thermocellum . This chapter describes the purification of the cellobiohydrolases I and II from trichoderma reesei and penicilliurn pinophilum and the purification of the Endocellulase C (EGC) from Clostridium thermocellum Cloned in E. coli .

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei.

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.

Structural changes in cellobiohydrolase I upon binding of a macromolecular ligand as evident by SAXS investigations

Xylan from Rhodymenia palmata binds to the cellobiohydrolase I from Trichoderma reesei (CBH I) or its core protein, inhibiting their activity. Adsorption onto microcrystalline cellulose (Avicel) is reduced approximately 30% for intact CBH I and nearly 50% for the core, whereas the effects with cellobiose are negligible. Structural changes concomitant with this binding are studied in solution by small angle X-ray scattering. In the tadpole structure typical for the CBH I [Abuja et al., 1988] the lengthening of the tail part is the most salient observation when xylan is present which accounts for an increase in Dmax (18.0 to 22.0 nm) and radius of gyration (4.74 to 5.18 nm). When xylan binds to the core the radius of gyration remains nearly unchanged. Here a model can be constructed showing a xylan molecule on the surface of the core protein near the tail part.