Mode of action of endoglucanase III from Trichoderma reesei

Abstract

Endoglucanase III (EG III) was purified to homogeneity from the culture medium of Trichoderma reesei QM 9414. It has a molecular mass of 48 kDa, and an isoelectric point of 5.1. Maximal activity was observed between pH4 and 5. Celloligosaccharides and their chromophoric derivatives were used as substrates, and the reaction products were analysed by quantitative h.p.l.c. Nucleophilic competition experiments (between methanol and water) allowed unequivocal assessment of cleavage sites. EG III preferentially released cellobiose (or the corresponding glycoside) from the reducing end of the higher cellodextrins. A putative binding model containing five subsites is proposed. The pH-dependence of 4'-methylumbelliferyl beta-cellotrioside hydrolysis indicates the presence of a protonated group with a pK 5.5 in the reaction mechanism, and the possible involvement of a carboxy group is corroborated by a temperature study (delta Hion = -15.9 J/mol). This, together with independent evidence from affinity-labelling experiments [Tomme, Macarrón and Claeyssens (1991) Cellulose '91, New Orleans, Abstr. 32] and n.m.r. studies [Gebbler, Gilkes, Claeyssens, Wilson, Béguin, Wakarchuk, Kilburn, Miller, Warren and Withers (1992) J. Biol. Chem. 267, 12559-12561], favours the assumption of a lysozyme-type (retention of configuration, two essential carboxy groups) mechanism for this family A cellulase.

Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris

“…Compared to the kinetic parameters reported by Macarron et al (1993) and Nakazawa et al (2009) for CMCase activity, the K cat value obtained here is higher in comparison to EGIII from T. reesei, with the K m being twice as high. The K m is similar to that found for endoglucanase activity in the crude extract of T. harzianum IOC-3844 (19 g/L) and the endoglucanase from T. harzianum ETS 323 (23 g/L) (de Castro et al, 2010a;Liu et al, 2010).…”

“…Glycosyl hydrolase family 12 comprises hydrolytic enzymes that cleave glycosidic bonds between two carbohydrates or a carbohydrate from a non-carbohydrate moiety. The cellulases from this family have no CBD and do not easily access crystalline cellulose (Macarron et al, 1993;Okada et al, 1998). However, these enzymes contribute to the breakdown of biomass by hydrolyzing amorphous cellulose and acting synergistically with expansin-like proteins (Arantes and Saddler, 2010).…”

Role of four major cellulases in triggering of cellulase gene expression by cellulose in Trichoderma reesei

“…However, the apparent role of egl2 has not yet been described. The particular efficacy of CBH II and EG II in the generation of the inducer is puzzling, since the products of their reactions are widely different (5,9,13). It is also noteworthy that neither CBH II nor EG II forms transglycosylation products, which have been reported as potent inducers of cellulase formation (23).…”

Cloning, Sequencing, and Expression of a Thermostable Cellulase Gene ofHumicola grisea

“…The optimal pH of EGL2 was 5.0, and EGL2 was stable at least in a range of pH 4.0-11.0 at 4°C for 20 h. EGL2 s·~emed a thermostable endoglucanase when it was compared with other fungal endoglucanases. 19,20) As we have not purified native EGL2 from H. grisea, the enzymatic properties of native EGL2 are unknown (endoglucanase 2 purified from H. grisea previously2) does not correspond to EGL2). However, the thermal stability of cloned EGL2~;eems to reflect that of native EGL2, as the genus Humicola has been known to produce several kind of thermostable cellulases.…”