Trichoderma reesei alpha-1,2-mannosidase: structural basis for the cleavage of four consecutive mannose residues.

The process of N-glycosylation of eukaryotic proteins involves a range of host enzymes that delete or add saccharide monomers. While endoplasmic reticulum (E.R.) mannosidases cleave only one mannose to produce the Man8B isomer, an alpha-1,2-mannosidase from Trichoderma reesei can sequentially cleave all four 1,2-linked mannose sugars from a Man(9)GlcNAc(2) oligosaccharide, a feature reminiscent of the activity of Golgi mannosidases. We now report the structure of the T. reesei enzyme at 2.37 A resolution. The enzyme folds as an (alpha alpha)(7) barrel. The substrate-binding site of the T. reesei mannosidase differs appreciably from the Saccharomyces cerevisiae enzyme. In the former, shorter loops at the surface allow substrate protein to come closer to the catalytic site. There is more internal space available, so that different oligosaccharide conformations are sterically allowed in the T. reesei alpha-1,2-mannosidase.     

Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides

A calcium-dependent alpha-1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides was purified to homogeneity from rabbit liver microsomes. N-terminal amino acid analysis was consistent with the presence of a homogeneous protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and nonreducing conditions, revealed a single protein band with an apparent molecular weight of 52,000. Gel filtration and sedimentation analysis under nondenaturing conditions suggested that the purified enzyme is a monomeric protein. The mannosidase is a glycoprotein based on the presence of protein-linked sugar and specific binding of the enzyme to concanavalin A-Sepharose. Purified mannosidase was optimally active between pH 5.0 and 6.0. The enzyme was inactive with p-nitrophenyl-alpha-D-mannopyranoside and was inhibited by deoxymannojirimycin but not by swainsonine. The enzyme was specifically activated by Ca2+, with half-maximal activation occurring at concentrations of 10 microM or less and was inhibited by Mn2+, Co2+, Ba2+, and Zn2+. Calcium ions protected the enzyme against inactivation by p-chloromercuribenzoate. Rabbit liver mannosidase hydrolyzed alpha-1,2-mannosyl-mannose linkages in a variety of substrates including methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (Schutzbach, J. S. (1987) Anal. Biochem. 167, 279-283), ovalbumin glycopeptide IV, and the high mannose chains of thyroglobulin and phytohemagglutinin-P. Approximately 70% of the alpha-1,2-linked mannosyl units in the oligosaccharides of thyroglobulin were accessible to rabbit liver alpha-mannosidase, whereas most of the alpha-1,2-mannosyl units in phytohemagglutinin were resistant to digestion prior to heat denaturation of the plant lectin.     

Cloning and expression of 1,2-alpha-mannosidase gene (fmanIB) from filamentous fungus Aspergillus oryzae: in vivo visualization of the FmanIBp-GFP fusion protein

1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-alpha-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.     

High Production of beta-Glucosidase in Schizophyllum commune: Isolation of the Enzyme and Effect of the Culture Filtrate on Cellulose Hydrolysis

Optimization experiments with response surface statistical analysis were performed with Schizophyllum commune to obtain high beta-glucosidase yields. The factors in the optimization experiment were the concentrations of cellulose, peptone, and KH(2)PO(4). Their optimal values were 3.2, 3.0, and 0.2 g/100 ml, respectively. Enzyme assays revealed very high beta-glucosidase (22.2 U/ml) and cellobiase (68.9 U/ml) yields. The avicelase yield was low as compared with that from Trichoderma reesei. Mixtures of S. commune and T. reesei culture filtrates caused faster and more extensive saccharification of Avicel than could be achieved by either filtrate alone. A beta-glucosidase was isolated and purified from the optimized culture filtrate of S. commune. The electrophoretic mobility of the purified beta-glucosidase indicated a molecular weight of 97,000. The amino acid composition was similar to that of beta-glucosidase from T. reesei. The acidic (aspartate and glutamate) residues or their amides or both made up approximately 20% of the protein. The NH(2)-terminal amino acid of the enzyme was histidine.     

Characterization of a novel alpha-D-mannosidase from rat brain microsomes

A new alpha-D-mannosidase has been identified in rat brain microsomes. The enzyme was purified 70-100-fold over the microsomal fraction by solubilization with Triton X-100, followed by ion exchange, concanavalin A-Sepharose, and hydroxylapatite chromatography. The purified enzyme is very active towards mannose-containing oligosaccharides and has a pH optimum of 6.0. Unlike rat liver endoplasmic reticulum alpha-D-mannosidase and both Golgi mannosidases IA and IB, which have substantial activity only towards alpha 1,2-linked mannosyl residues, the brain enzyme readily cleaves alpha 1,2-, alpha 1,3-, and alpha 1,6-linked mannosyl residues present in high mannose oligosaccharides. The brain enzyme is also different from liver Golgi mannosidase II in that it hydrolyzes (Man)5GlcNAc and (Man)4GlcNAc without their prior N-acetylglucosaminylation. Moreover, the facts that the ability of the enzyme to cleave GlcNAc(Man)5GlcNAc, the biological substrate for Golgi mannosidase II, is not inhibited by swainsonine, and that p-nitrophenyl alpha-D-mannoside is a poor substrate provide further evidence for major differences between the brain enzyme and mannosidase II. Inactivation studies and the co-purification of activities towards various substrates suggest that a single enzyme is responsible for all the activities found. In view of these results, it seems possible that, in rat brain, a single mannosidase cleaves asparagine-linked high mannose oligosaccharide to form the core Man3GlcNAc2 moiety, which would then be modified by various glycosyl transferases to form complex type glycoproteins.     

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.     

Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei

A novel fungal beta-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These beta-glucosidases show significant homology to plant beta-glucosidases belonging to the beta-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant beta-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to beta-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of beta-galactosidase activity, while recombinant T. reesei BGL2 showed weak beta-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.     

人溶酶体α-甘露糖苷酶cDNA的克隆与表达

溶酶体α-甘露糖苷酶是糖蛋白降解途径的主要外糖苷酶,该酶缺陷引起溶酶体α-甘露糖苷贮积症.用RT-PCR法从HeLa细胞中克隆的人溶酶体α-甘露糖苷酶cDNA含有1个由2964bp组成的阅读框,编码由988个氨基酸组成的多肽,前26个氨基酸为潜在的前导序列,成熟多肽的预测分子量为111kD,具有11个潜在的N-交联糖基化部位.用逆转录病毒介导法导入患者细胞后,该cDNA表达高活性α-甘露糖苷酶.序列分析表明,此cDNA与已发表的拟似人溶酶体α-甘露糖苷酶cDNA有不同程度的差异,尤其是第2315位碱基的T-C转换可能与控制酶活性有关     

A fluorescence assay for alpha-1,2-mannosidases involved in glycoprotein processing reactions

A highly specific, sensitive, and convenient fluorescence assay for alpha-1,2-mannosidases involved in glycoprotein processing reactions is described. The assay utilizes a coupled enzyme system to determine the amount of free mannose liberated from the disaccharide O-methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside by the alpha-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated alpha-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the alpha-1,2 linkage. The mannosyl linkages in alpha-1,3- and alpha-1,6-linked methyl-disaccharides, in methyl-alpha-D-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the alpha-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The Km for the alpha-1,2-linked methyl-disaccharide is 0.5 mM.     

Purification and Properties of a Glycoprotein Processing alpha-Mannosidase from Mung Bean Seedlings

The microsomal fraction of mung bean seedlings contains mannosidase activities capable of hydrolyzing [³H]mannose from the [³H]Man₉GlcNAc as well as for releasing mannose from p-nitrophenyl-α-d-mannopyranoside. The glycoprotein processing mannosidase was solubilized from the microsomes with 1.5% Triton X-100 and was purified 130-fold by conventional methods and also by affinity chromatography on mannan-Sepharose and mannosamine-Sepharose. The final enzyme preparation contained a trace of aryl-mannosidase, but this activity was inhibited by swainsonine whereas the processing enzyme was not. The pH optimum for the processing enzyme was 5.5 to 6.0, and activity was optimum in the presence of 0.1% Triton X-100. The enzyme was inhibited by ethylenediaminetetraacetate while Ca²⁺ was the most effective cation for reversing this inhibition. Mn²⁺ was considerably less effective than Ca²⁺ and Mg²⁺ was without effect. The processing mannosidase was inhibited by α1,2- and α1,3-linked mannose oligosaccharides (50% inhibition at 3 millimolar), whereas free mannose and α1,6-linked mannose oligosaccharides were ineffective. Mannosamine was also an inhibitor of this enzyme. The aryl-mannosidase and the processing mannosidase could also be distinguished by their susceptibility to various processing inhibitors. The aryl-mannosidase was inhibited by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol but not by deoxymannojirimycin or other inhibitors, while the processing mannosidase was only inhibited by deoxymannojirimycin. The processing mannosidase was incubated for long periods with [³H]Man₉GlcNAc and the products were identified by gel filtration. Even after a 24 hour incubation, the only two radioactive products were Man₅GlcNAc and free mannose. Thus, this enzyme appears to be similar to the animal processing enzyme, mannosidase I, and is apparently a specific α1,2-mannosidase.     

Optimization of ellagic acid production from ellagitannins by co-culture and correlation between its yield and activities of relevant enzymes

Aspergillus oryzae was co-cultured with Trichoderma reesei using acorn cups extract containing up to 62% ellagitannins as substrate to produce ellagic acid with relatively high levels of ellagitannin acyl hydrolase, cellulase and xylanase. Ellagitannins concentration, initial pH, T. reesei and A. oryzae during the fermentation were identified as important process parameters effecting ellagic acid accumulation and the enzymes syntheses. These parameters were optimized by uniformity design to determine the optimum condition for ellagic acid production. Under optimum operational condition, ellagic acid yield could be arrived at 24%, when the fermentation run lasted 96h with an initial pH of 4.5, an ellagitannins concentration of 4gl(-1), T. reesei of 3ml and A. oryzae of 3ml. Meanwhile, it was found that the three enzymes activities correlated very well with ellagic acid yield, resulting in model with high coefficient of determination (R(2)=0.98). The results indicate that the mixed culture of T. reesei and A. oryzae is an effective approach to produce an enzyme system of degrading ellagitannins for ellagic acid production.     

Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties.

Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.     

Cloning of a gene encoding hydroxyquinol 1,2-dioxygenase that catalyzes both intradiol and extradiol ring cleavage of catechol.

Two Escherichia coli transformants with catechol 1,2-dioxygenase activity were selected from a gene library of the benzamide-assimilating bacterium Arthrobacter species strain BA-5-17, which produces four catechol 1,2-dioxygenase isozymes. A DNA fragment isolated from one transformant contained a complete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme expressed by the ORF was purified to homogeneity and characterized. When hydroxyquinol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate specificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a novel dioxygenase that catalyzed both the intradiol and extradiol cleavage of catechol.     

Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.     

Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.     

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.     

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.     

Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.