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PubMed1976

Poly(A)-associated RNA from the mitochondrial fraction of the fungus Trichoderma.

Total RNA was extracted from purified mitochondrial and cytoplasmic fractions of germinating conidia of Trichoderma viride and bound to oligo(dT)-cellulose at 22 and 4 degrees C. Under chromatographic conditions which retained very short poly(A) segments (i.e., 4 degrees C), up to 10% of short-term 32PO4-lebeled RNA from the mitochondrial fraction were selectively bound. The poly(A)-associated RNAs from the mitochondrial and cytoplasmic fractions showed the following characteristics. (a) On polyacrylamide gels mitochondrial fraction RNA had a distinctive pattern with a major peak at about 22 S and a smaller one at about 29 S; in contrast, cytoplasmic fraction RNA was heterogenously distributed along the gel. (b) The poly(A) segment released by RNAase digestion of mitochondrial fraction poly(A)-associated RNA migrated on polyacrylamide gels as molecules 20-25-nucleotides long, while that of the cytoplasmic fraction showed an apparent size of 50-60 nucleotides. (c) Mitochondrial fraction RNA bound to oligo(dT)-cellulose in the cold had a guanine + cytosine content of 21% versus 34% for bulk mitochondrial RNA and 48% for cytoplasmic poly(A)-associated RNA; the oligo(dT)-bound RNAs were further identified by their high percentages of adenine residues (46% for the mitochondria and 30% for the cytoplasm). (d) The poly(A)-associated RNA fraction was translated, in vitro, in a cell-free protein-synthesizing system from wheat germ. The products induced by cytoplasmic RNA showed a complex pattern on polyacrylamide gels of many polypeptides ranging in molecular weights from 10000 to 40000. The pattern induced by mitochondrial fraction RNA however, was much simpler, revealing two discrete, main products: a major one at Mr approximately 13000 and a minor one at Mr approximately 20000.

Chromatography, Affinity↗

PubMed1985

The cellulase of Trichoderma viride. Purification, characterization and comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases.

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.

Cellulase↗

PubMed1985

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II.

The 1,4-beta-glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4-methylumbelliferyl beta-D-glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n = 3 - 5]. The reaction has been followed by quantitative high-performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 +/- 0.2) min-1 for MeUmb(Glc)3 and (9 +/- 2) min-1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn-over numbers (17 +/- 4) min-1 and (21 +/- 6) min-1, respectively. Methylumbelliferyl beta-glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr = 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2 degrees C and 20 degrees C. The association constants fitted linear van't Hoff plots in both cases. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non-chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl beta-D-glucopyranoside and cellobiose as ligands also point at an extended binding site.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1987

Purification and characterization of a beta-glucosidase from Trichoderma reesei.

A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.

Amino Acids↗

PubMed1987

Beta-glucosidase from Trichoderma reesei. Substrate-binding region and mode of action on [1-3H]cello-oligosaccharides.

To determine the mode of action of the beta-glucosidase from Trichoderma reesei a method was developed for synthesizing [1-3H]cello-oligosaccharides with specific radioactivities of approximately 3000 Ci/mol. The beta-glucosidase removed glucosyl residues from the non-reducing end of the [1-3H]cello-oligosaccharides in a multiple attack mode with little tendency to attack the substrates repetitively. Values of Km were lower for longer cello-oligosaccharides, whereas values of V remained essentially constant. A subsite map, constructed using values of V/Km for the cello-oligosaccharides, showed that the substrate-binding region comprises primarily three subsites.

Binding Sites↗

PubMed1989

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Binding of small ligands to the 1,4-beta-glucan cellobiohydrolase II and influence of glucose on their affinity.

Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein-difference-absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped-flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329-334] indicating an extended active center, with putative subsites ABCD. Subsite A specifically complexes with beta-D-glucosides and D-glucose; at 25 degrees C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4-37 degrees C) was characterized by a substantial increase of the enthalpic term (delta delta H = -35 kJ mol-1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4-methylumbelliferyl beta-D-cellobioside by one order of magnitude: k+ = (3.6 +/- 0.5) x 10(-5) M-1 s-1 versus (5.1 +/- 0.1) x 10(-6) M-1 s-1 (in the absence of glucose) and k- = (1.3 +/- 0.1) s-1 versus (14.0 +/- 0.3) s-1. As deduced from substrate-specificity studies and inhibition experiments, subsite B interacts with terminal non-reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D. Association constants 10-100 times higher than those for cellobiose or its glycosides were observed for D-glucopyranosyl-(1----4)-beta-D-xylopyranose and cellobionolactone derivatives, suggesting 'transition-state'-type binding for these ligands at subsite C. Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (-34 kJ mol-1) as compared to cellobiose (-28.3 kJ mol-1) and MeUmb(Glc)2 (-11.6 kJ mol-1). This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.

Binding Sites↗

PubMed1990

Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii.

The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic-acid-producing strain of Trichoderma koningii M3947 was shown to contain the koningic-acid-resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone-rich medium, however, no koningic acid production was observed, and the koningic-acid-sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 x 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent Km values of GAPDH I and II for glyceraldehyde 3-phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.

Amino Acid Sequence↗

PubMed1991

Tricholongins BI and BII, 19-residue peptaibols from Trichoderma longibrachiatum. Solution structure from two-dimensional NMR spectroscopy.

Two nonadecapeptides, tricholongins BI and BII, which display antifungal and antibacterial activities, have been isolated from in vitro cultures of the fungus Trichoderma longibrachiatum. The peptides were separated by reversed-phase HPLC; their amino acid compositions were determined by gas chromatography and their sequences by positive-ion fast-atom-bombardment mass spectrometry and high-field NMR. These linear peptides, containing mainly hydrophobic L-amino acids, 8-9 2-aminoisobutyric acid residues and exhibiting an acetylated N-terminal residue and an amino alcohol C-terminal leucinol belong to the peptaibol class. The methanol solution structure of tricholongins BI and BII has been investigated using both one- and two-dimensional NMR techniques. The total 1H-NMR and 13C-NMR assignments are given. By a combination of the 3JNH,C alpha H coupling constant values, temperature coefficients of the NH and CO groups, amide hydrogen/deuterium-exchange rate measurements and NOE data, a secondary structure for tricholongins in solution has been proposed. Both peptides adopt a similar alpha-helical conformation with a hinge around Pro13 resulting from two 3(10) bonds. The results suggest that the N-terminus contains mixed alpha/3(10) bonds. The membrane permeability modifications induced by tricholongins have been assayed by the use of liposomes composed of egg phosphatidylcholine with 20-30% cholesterol. The peptide-induced leakage of an entrapped fluorescent probe has been followed by fluorescence spectroscopy. In a concentration range of 0.13-0.31 microM, tricholongins induce the leakage of 50% of the entrapped material in 20 min.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of two xylanases from Trichoderma longibrachiatum.

Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl-cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while xylobiose appeared to inhibit xylanase B, but not xylanase A.

Chromatography, Gel↗

PubMed1991

The endo-1,4-beta-glucanase I from Trichoderma reesei. Action on beta-1,4-oligomers and polymers derived from D-glucose and D-xylose.

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.

Cellulase↗

PubMed1992

Isolation and characterization of three chitinases from Trichoderma harzianum.

Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n = 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n = 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross-reaction, suggesting that each protein is encoded by a different gene.

Carbohydrate Conformation↗

PubMed1995

Kinetic and stereochemical studies of manno-oligosaccharide hydrolysis catalysed by beta-mannanases from Trichoderma reesei.

The two beta-mannanases from Trichoderma reesei with pI of 4.6 and 5.4, respectively, have been characterised by NMR spectroscopy. Following the kinetics of manno-oligosaccharide degradation with complete progress-curve analysis the stereospecificity and degradation pattern have been delineated. It was found that degradation of mannotriose and mannopentaose proceeds with retention of the anomeric configuration. Mannotriose degradation proceeds by almost random release of mannose. For mannopentaose there is initially no mannose formed showing that only the two middle mannosidic linkages are attacked. Progress-curve analysis shows that there is preference (70%) for cleavage of mannopentaose in such a way that mannobiose is released from the reducing end. The final product composition from the mannotriose degradation showed that transglycosylation has to be taken into account. Model calculation and progress-curve analysis showed that the transglycosylation rate is the fastest of all the rates in this system, 15 s-1 compared with mannohexaose and mannotetraose hydrolysis rates of 2 s-1 and mannotriose hydrolysis rate of 0.03 s-1 at 50 degrees C.

Catalysis↗

PubMed1995

Progress-curve analysis shows that glucose inhibits the cellotriose hydrolysis catalysed by cellobiohydrolase II from Trichoderma reesei.

NMR spectroscopy and HPLC were used to investigate the hydrolysis of cellotriose by cellobiohydrolase II from Trichoderma reesei. Substrate and product concentrations were followed as a function of time. Progress curves were calculated by forward numerical integration of the full kinetic equations and were fitted to the experimental data. Binding and rate constants were obtained from this fit, whereby no initial slope or Michaelis-Menten approximation was used. The progress curves from a single experiment sufficed to produce agreement with the Michaelis-Menten model (eight experiments). The absence of a kinetic isotope effect was proven. The progress-curve analysis showed that a simple degradation model cannot describe the experimental time-courses at substrate concentrations greater than 1 mM. A model containing competitive inhibition from cellobiose as well as non-competitive inhibition from glucose was developed. This four-parameter model accurately reproduces about 1000 experimental data points covering five orders of magnitude in oligosaccharide concentrations. Glucose binding to the enzyme/cellotriose complex retards, in a non-competitive fashion, cellotriose hydrolysis by at least a factor of 30. A structural model for the non-competitive inhibition is discussed. The NMR experiment also produced individual progress curves for the alpha and beta anomers. The beta anomer of cellotriose was degraded 2.5-times faster than the alpha anomer.

Catalysis↗

PubMed1996

Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI.

The hfb1 gene of the filamentous fungus Trichoderma reesei, previously cloned as a gene which was abundantly expressed when the fungus was grown on glucose-containing medium, was shown to encode a novel fungal hydrophobin. The encoded 97-amino-acid protein is cysteine-rich and has a typical signal sequence for secretion. Signal-sequence cleavage and putative proteolytic processing results in the mature HFBI protein of 75 amino acids. Antibodies raised against the HFBI protein expressed in Escherichia coli detected the T. reesei HFBI protein in the fungal cell wall and in the culture medium of submerged glucose-containing cultures. The identity of HFBI was verified by N-terminal and peptide sequencing of proteins purified both from the cell wall and culture medium. In the cell wall most of the HFBI formed SDS-insoluble complexes that could be extracted with trifluoroacetic acid. Bubbling or freezing of the culture medium caused HFBI to form aggregates that coprecipitated with a yellow pigment produced by the fungus.

Amino Acid Sequence↗

PubMed1996

Three alpha-galactosidase genes of Trichoderma reesei cloned by expression in yeast.

Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside. The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T. reesei. AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T. reesei and a beta-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T. reesei.

Amino Acid Sequence↗

PubMed1996

Acetyl xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding domain.

The axe1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe1 product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.

Acetylesterase↗

PubMed1996

Cello-oligosaccharide hydrolysis by cellobiohydrolase II from Trichoderma reesei. Association and rate constants derived from an analysis of progress curves.

The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1-12 s-1 at 27 degrees C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for collotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.

Binding Sites↗

PubMed1997

Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei--cloning and characterization of the hfb2 gene.

The hfb2 gene encoding the hydrophobin HFBII of the filamentous fungus Trichoderma reesei was isolated by heterologous hybridization using the vegetative hydrophobin I, hfb1, gene of T. reesei as a probe. The hfb2 gene codes for a typical fungal secreted hydrophobin of 71 amino acids containing eight cysteine residues. The amino acid similarity towards HFBI is 69%. The HFBII protein was isolated from the fungal spores by extraction with trifluoroacetic acid/acetonitrile solution, and by bubbling from the lactose-based culture medium. Expression of the hfb1 and hfb2 genes is divergent. hfb1 expression was only observed in vegetative cultures on glucose-containing and sorbitol-containing media. It was not expressed on media containing complex plant polysaccharides, cellulose, xylan, cellobiose or lactose, whereas hfb2 was highly expressed in vegetative cultures on these media. Expression of hfb2 was also strongly induced by N and C starvation, by light and in conidiating cultures.

Amino Acid Sequence↗