Marc Claeyssens

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.

The use of 4-methylumbelliferyl and other chromophoric glycosides in the study of cellulolytic enzymes

HPLC‐analysis of the reaction products of a series of 4‐methylumbelliferyl glycosides from cello‐oligosaccharides, used as substrates of a cellobiohydrolase from Trichoderma reesei, proves the lack of specificity for terminal cellobiosyl groups. Also, different reaction patterns are observed for this CBHI and for an endocellulase, when acting on these same substrates. 4‐Methylumbelliferyl β‐D‐lactoside is an unexpected substrate for CBHI, yielding only lactose and phenol as reaction products. The binding characteristics of p‐nitrobenzyl 1‐thio‐β‐D‐lactoside for this enzyme are determined by a dia‐filtration technique, yielding 1 binding site and an association constant of 4.0 × 104 M−1.

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from Trichoderma reesei

BACKGROUND Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

Engineering the Exo-loop of Trichoderma reesei Cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

Degradation of Xylan to d-Xylose by Recombinant Saccharomyces cerevisiae Coexpressing the Aspergillus niger β-Xylosidase (xlnD) and the Trichoderma reesei Xylanase II (xyn2) Genes

ABSTRACT The β-xylosidase-encoding xlnD gene ofAspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor α1 signal peptide (MFα1s) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant β-xylosidase showed optimum activity at 60°C and pH 3.2 and optimum stability at 50°C. The Ki(app) value ford-xylose and xylobiose for the recombinant β-xylosidase was determined to be 8.33 and 6.41 mM, respectively. TheXLO2 fusion gene and the XYN2 β-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of β-xylanase activity when expressing only the β-xylanase and 860 nkat/ml when coexpressing the β-xylanase with the β-xylosidase. The maximum β-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the β-xylanase. Coproduction of the β-xylanase and β-xylosidase enabled S. cerevisiae to degrade birchwood xylan tod-xylose.

Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates

The 4‐methylumbelliferyl β‐D‐glycosides of glucose, cellobiose, cellotriose and lactose are used to differentiate several exo‐cellobiohydrolase, endocellulase and β‐glucosidase activities in crude cellulase from Trichoderma reesei. Spectrophotometric or fluorimetric assays allow simple detection and quantitative measurements of eluate activities from ion‐exchange chromatography and after analytical gel electrofocusing. Using the fluorophoric glucoside several β‐glucosidases can be visualised after isoelectric focusing on polyacrylamide gels. The use of the lactoside and of the same substrate supplemented with cellobiose as inhibitor allows a clearcut distinction to be made between endocellulase II and exo‐cellobiohydrolase I. Both enzymes are present as ‘iso‐enzyme’ mixtures. With the cellotrioside only one fraction is detectable (endocellulase III). The same methods could be used in culture growth experiments.

The endo-1,4-β-glucanase I from Trichoderma reesei

The reaction mechanism of the non-specific endo-1,4-beta-glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing-end3H-labelled and universally 14C-labelled cellooligosaccharides, as well as reducing-end3H-labelled xylooligosaccharides. The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible. During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive D-glucose or cellobiose. The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize cellooligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of D-glucosyl and D-xylosyl residues. The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, -II and +II show the highest affinities, 16.7 kJ.mol-1 and 7.1 kJ.mol-1, respectively.

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-d-mannosidase

Abstract A cDNA encoding 1,2-α- d -mannosidase mds1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56 266 Da and shows high similarity to the amino acid sequences of 1,2-α- d -mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris . Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae α-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analysed. The enzyme was characterized as a class-I mannosidase.

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii.

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.

Fluorogenic and chromogenic glycosides as substrates and ligands of carbohydrases

Publisher Summary Nitrophenylglycosides are frequently used substrates of carbohydrases. Alternatively, the 4-methylumbelliferyl (7-hydroxy-4-methylcoumaryl) derivatives offer a more sensitive (fluorometric) method of detection. Some of these compounds have become commercially available. This chapter describes the preparation and use of these glycosides in the study of some cellulolytic enzymes. The difference in spectral properties of free 4-methylumbelliferone and its carbohydrate conjugates allows sensitive and continuous assays of cellulolytic activities in absorption or fluorescence modes. The preparation and use of 1-thio derivatives with different chromophoric reporter groups are included. Due to their optical characteristics, the chromophoric (fluorochromic) derivatives offer distinct advantages over the use of classical substrates of cellulolytic enzymes, as they are sensitive tools for the determination of the number of binding sites, study of association modes, and binding kinetics, breakdown patterns, and inhibition characteristics. The chapter describes some applications of chromophoric.