3Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia; 4VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044, VTT Finland; and 5Proteome Systems Ltd, Locked Bag 2073, North Ryde, NSW 1670, Australia
Received on November 1, 2001; revised on January 17, 2002; accepted on January 24, 2002.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: acetylxylan esterase/glycosylation/hemicellulase/isoforms/Trichoderma reesei
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Even though most fungal hydrolases are glycoproteins of considerable biotechnical interest, limited information is available on the sites, type, and composition of glycosylation on these enzymes. In general, the fungal N-linked glycan core has shown to be identical to the mammalian N-linked core (Man3GlcNAc2). However, the occurrence of single N-acetyl-glucosamine reported on the main cellobiohydrolase I (CBHI) of T. reesei ALKO2877 and QM9414 (Harrison et al., 1998; Klarskov et al., 1997
) suggests that strains of T. reesei N-glycosylate CBHI differently. Structural characterization and studies into the effects of N-linked carbohydrate chains of different enzymes, such as
-galactosidase (Savel'ev et al., 1997
) and cellobiohydrolase I from T. reesei (Maras et al., 1997
), glucoamylase from A. awamori X 100/D27 (Eriksen et al., 1998
), and
-amylase from A. awamori (Chen et al., 1994
) have been carried out. The importance of N-linked glycosylation for secretion or stability of extracellular enzymes from filamentous fungi appears to differ between fungi (Neustroev et al., 1993
; Eriksen et al., 1998
; Chen et al., 1994
).
O-linked glycans of glucoamylase from A. awamori (Neustroev et al., 1993), Aspergillus niger (Gunnarsson et al., 1984
) and CBHI of T. reesei (Harrison et al., 1998
) include di- and trisaccharides containing terminal glucose, mannose, and galactose. In CBHI from T. reesei, a glucose residue has been found to be directly linked to the polypeptide chain (Gum and Brown, 1976
). Analysis of stability changes following decreased glycosylation of A. awamori linker region (Neustroev et al., 1993
) suggests that O-linked sugars essentially contribute to the stabilization of glucoamylase. Furthermore, the linker glycopeptide seems to stabilize the binding domain against reversible thermal and chemical denaturation (Williamson et al., 1992
). With respect to the diversity of sugar residues present in the glycoproteins, filamentous fungi bear greater similarity to mammalian cells than yeast, which typically hypermannosylate. Apart from O-and N-linked glycosylation, further structural diversification may occur by covalent attachment of phosphate, sulfate, acetyl, or methyl groups to the sugar (reviewed in Lis and Sharon, 1993
).
Previous partial characterization of AXE of T. reesei RutC-30 has shown that the enzyme is modified by both N- and O-linked sugars comprising up to 1215% by weight of the molecule (Margolles-Clark et al., 1996; Sundberg and Poutanen, 1991
). However, the exact nature of the glycosylation of the acetyl xylan esterase of T. reesei has not been resolved. Characterization of the N- and O-linked carbohydrates of AXE presented in this article will contribute to the understanding of the effect of posttranslational modifications on fungal hydrolytic enzymes. Composition, positional information of O-linked sugars on the peptide, nature of oligosaccharide heterogeneity, and modifications of glycosylation of the purified glycoprotein AXE from T. reesei Rut-C 30 will be discussed. In a wider perspective, in the production of heterologous glycoproteins in filamentous fungi, especially those of therapeutic importance, a good knowledge of the nature of the glycans produced by the host is elementary.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Papain digestion of AXE produced two peptides, amino acids pyrGlu1Gly215, which constitute the catalytic core domain, and amino acids Gly216Leu271, constituting the linker and SBDs. The linker-SBD peptide was found to be O-glycosylated with hexose residues and modified by sulfate. Rather than simply releasing the O-glycans and obtaining average structural information, the density of O-glycosylation on the linker peptide was determined using a modified Edman amino acid sequencing technique. This approach showed not only the sites that were occupied but also that each theonine and serine was heterogeneously glycosylated with at least one or two hexoses. Although evidence of the attachment site of sulfation is unknown, we presume that it is most likely attached to one of the O-glycans present in the linker region by extrapolation to the CBHI glycopeptide linker. This peptide is sulfated in the T. reesei strain ALKO2877 but possesses no free hydroxy-amino acids or tyrosine within the linker to accommodate a sulfate group (Harrison et al., 1998). All of the oligosaccharide masses of N-linked glycans liberated by PNGaseF treatment corresponded to the oligosaccharide composition HexNAc2Hex37PNa, which describes a typical N-linked core of HexNAc2Hex3, plus up to four additional hexoses and phosphate.
Monosaccharide analyses of the whole intact AXE protein revealed the presence of mannose, galactose, glucose, and N-acetlyglucosamine. Analysis of the linker-SBD peptide in isolation revealed only mannose, galactose, and glucose in a ratio supporting the view that N-acetylglucosamine derives from the sole N-glycan attached to the core peptide. It was not possible to derive a monosaccharide composition for the core peptide, due to the coelution of the core peptide and native AXE over reversed-phase C8 and gel-filtration columns. The observed average ratio of sugars for the linker-SBD peptide in monosaccharide analysis was 16.9:1 (moles of sugar per mole of peptide, excluding 4.4 moles of glucose) and is consistent with the observation that the most intense isoform of the linker peptide possessed 16 hexoses. It is difficult to verify the true presence of glucose in hydrolysis-based monosaccharide analyses becaus glucose is often viewed as a contaminant. Others (Maras et al., 1997; Gunnarsson et al., 1984
; Takayanagi et al., 1992
) have confirmed the presence of N- and O-linked glucose on Aspergillus and Trichoderma glycoproteins by several techniques, including nuclear magnetic resonance, and given the high amount of glucose observed in this report, we believe that glucose is likely to be also present on AXE. The presence of mannose, galactose, and probably glucose on the AXE linker is in agreement with that observed by us for another glycosylhydrolase, T. reesei CBHI (Nevalainen et al., 1997
; Harrison et al., 1998
).
A previous report on the characterization of the AXE glycoprotein observed two protein activities with pIs of 6.8 and 7.0 by gel electrophoresis (Poutanen et al., 1990), compared to the three more acidic spots of pI 4.90, 5.25, and 5.98 observed in this report. Discrepancy between our results may be due to the nature of carrier ampholytes used in the previous report or difference in cultivation conditions, which may have an effect on the number of AXE forms detected. Given that the theoretical pI value of the published sequence of AXE obtained from SWISS-PROT (www.expasy.ch/sprot) is 5.56, and that the presence of sulfation and/or sulfation decrease the pI, it is probable that the most basic spot of 5.98 indeed represents the native AXE protein sequence without further modification. The three pI isomers of AXE observed in this report on 2D gels are consistent with our identification of two heterogeneous, charged modifications. Assuming that the overall contribution of phosphorylation and sulfation to the (decrease in the) pI of the full AXE protein is approximately equal within the effective pH resolution limits of the 2D gels used, and that the presence or absence of N-glycan phosphorylation occurs independently of the presence or absence of linker sulfation, it would be expected that AXE would resolve into three spots. From these, the center spot, comprising a mixture of protein molecules modified either by phosphorylation or sulfation, would be the most intense.
Two-dimensional PAGE of a complete papain digestion of AXE produced a pI isoform doublet of approximately 25 kDa apparent molecular mass, corresponding in mass to the catalytic core peptide; a second pI doublet of about 13 kDa apparent molecular mass, corresponding in mass to the linker peptide; and a fifth spot of about 22 kDa, corresponding to the apparent molecular mass of the protease papain. The observation of two spots for each of the linker-SBD and core peptides is consistent with the identification of partial sulfation of the linker and phosphorylation of the N-linked glycan on the core peptide. The large separation of the linker peptide spots in the charge dimension is likely due to the very large effect of the presence/absence of sulfation on such a small peptide because of its accordingly low buffering capacity.
Sulfation on a linker peptide and presence of phosphorylated N-glycans has been reported for T. reesei CBHI (Nevalainen et al., 1997; Harrison et al., 1998
). In the absence of further information concerning the biological/enzymatic differences between the differently modified enzymes in the different strains of T. reesei, the biological significance of these modifications is unclear. However, both modifications (phosphorylated N-glycans and sulfated O-glycans) have been implicated to some extent in organisms such as Leishmania and Dictyostelium in protein targeting and secretion (Haynes, 1998
). Sulfated carbohydrates have also been shown to play specific roles in well-defined biological processes, such as control of the circulatory half-life of lutenizing hormone, symbiotic interactions between leguminous plants and nitrogen-fixing bacteria, and homing of lymphocytes to lymph nodes (reviewed in Hooper et al., 1996
). Therefore, it is possible that the addition of a sulfate moiety turns a relatively common structure into a unique carbohydrate with the potential to be recognized by a specific receptor molecule. We have shown that the linker region of the filamentous fungal glycosylhydrolase AXE is heavily and heterogeneously O-glycosylated and possesses an unusual charged modification, sulfate. It is not unreasonable to presume that these perform some as-yet-unidentified biologically significant function.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteolytic digestion by papain
Papain digestion of AXE has been previously reported by Margolles-Clark et al. (1996). A slightly different protocol was used in the present study. Two hundred micrograms of AXE in 200 µl of 100 mM ammonium acetate, pH 8.0, was incubated with 8 µl of papain (1.4 mg/ml in 0.2 M phosphate buffer, 5 mM L-cysteine, 2 mM ethylenediamine tetra-acetic acid, pH 7.0) at 37°C for 120 min. A previous analytical scale experiment performed over a time-course of 30, 60, 120, and 240 min revealed that digestion of AXE into core and linker-SBD peptides was essentially complete at 120 min. Digested material was analyzed by MALDI-TOF and LC-MS. Analysis of core and linker peptides from this stock were performed by LC-MS (with a 1:20 split of the postcolumn flow to the MS), or by HPLC using the same LC method as described.
PNGaseF deglycosylation of AXE
PNGaseF digests followed the PNGase-F (Bio-Rad) denaturing protocol advised by the manufacturer. Twenty-five microliters of a solution of 0.6 mg/ml AXE (15 µg) in 100 mM ammonium bicarbonate, pH 8.0, was denatured by boiling for 5 min with 25 µl of 2x reaction buffer (Bio-Rad; 100 mM sodium phosphate, pH 7.5), and 2.5 µl of denaturing solution (2% SDS, 1 M ß-mercaptoethanol). The solution was cooled on ice prior to the addition of 2.5 µl of NP-40 (15%) and 4 µl of PNGaseF (2.5 U/ml in 20 mM Tris, pH 7.5, 50 mM NaCl, 1 mM ethylenediamine tetra-acetic acid). The digest was performed for 14 h at 37°C. The digest was verified by a shift in the apparent molecular weight of the native versus the PNGaseF-treated form on SDSPAGE and the released N-linked oligosaccharides were subsequently analysed by graphitized carbon LC-MS as described.
HPLC electrospray ionization TOF-MS (LC-MS)
Enzyme digests were routinely analyzed or preparatively purified by liquid chromatography on a SMART HPLC (Pharmacia). The instrument was fitted with either a SephasilTM C8 reversed-phase column (100 mm x 2.1 mm) for protease digests, or graphitized carbon cartridge (HyperCarb 10 mm x 4 mm, Shandon, Cheshire, UK) for oligosaccharide separations. For peptide separation, a linear gradient of 0.1% formic acid to 90% acetonitrile, 0.1% formic acid at a flow rate of 100 µl/min was routinely used, with separations proceeding over the time shown for each result. Typically, this was 15 min, though the resolution of sulfated/unsulfated forms was performed over 90 min. Separation of carbohydrates was performed over graphitized carbon from 0.1% formic acid to 25% acetonitrile, 50% butanol-saturated water, 0.1% formic acid at a flow rate of 100 µl/ min as a modification of chromatography previously reported (Packer et al., 1998). In both cases, postcolumn flow was directed to an electrospray ionization TOF mass spectrometer (Micromass, UK), except for preparative purifications of the core and linker peptides by HPLC in the absence of a mass spectrometer, in which case postcolumn flow was fraction-collected and the identity of fractions confirmed by direct injection into the mass spectrometer. Spectra were routinely acquired in the positive ion mode using two alternating scan functions, which differed only with respect to the voltage on the cone. Each of the "low" cone-voltage (30 V) and "high" cone-voltage scans (80 V) were acquired into separate files at a rate of 1 scan/s over the m/z ranges given in the results. Typically, these were m/z 50 to m/z 3000 for protein analyses, m/z 50 to m/z 1000 for O-linked carbohydrates, and m/z 50 to m/z 2500 for N-linked carbohydrates. Spectra in this report are presented without background subtraction or smoothing.
MALDI-TOF MS
MALDI-TOF MS spectra of papain digests of AXE were acquired on a Voyager DE-STR (Perseptive BioSystems, Framingham, MA) delayed-extraction, time-of-flight/reflectron instrument and/or a TofSpec2E (Micromass) delayed-extraction, time-of-flight/reflectron instrument. Samples were prepared on stainless steel or gold 96-well target plates by the dried-droplet method and allowed to air-dry at room temperature. Generally, this involved 0.5 µl of analyte with 0.5 µl of freshly prepared matrix solution; generally 10 mg/ml sinapinic acid in 50% acetonitrile, 0.1% TFA. Spectra were externally calibrated to reference spectra of myoglobulin and/or trypsin. Spectra are presented without background subtraction or smoothing.
Monosaccharide analysis
Monosaccharide compositional analyses were performed by both TFA or HCl acid hydrolysis and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Lyophilized protein samples of between 300750 pmol in screw cap Eppendorf tubes were resuspended in 50 µl of either 2 M TFA or 4 M HCl and hydrolyzed at 100°C for 4 h. Hydrolyzed samples were then dried in a Savant Speed-Vac and resuspended in 50 µl of water containing 0.5 mol 2-deoxy-D-glucose as internal standard. Released monosaccharides were quantitated by chromatography on a Dionex HPAEC-PAD system fitted with a Dionex CarboPac PA10 column (250 mm x 4 mm). Separations were performed isocratically with 18% NaOH at 1 ml/min over 20 min, with between run washes of 0.4 M NaOH for 20 min. Amounts are presented from concurrently performed TFA (for glucose, mannose, and galactose) and HCl (for glucosamine and galactosamine) hydrolyses.
2D PAGE
IPG strips (7 cm; pH 310 or pH 47 linear) were rehydrated with 5 µl of Orange G and 120 µl of sample solution (8 M urea, 4% CHAPS, 2 mM tributylphosphine, 40 mM Tris-base, 0.5% ampholytes, pH 9.5) for 67 h in covered rehydration trays (Bio-Rad). Reduced samples (2% dithiothreitol) in sample buffer, 1 µl bromophenol blue, 100°C, 5 min) of 3050 µl in volume were loaded in cups and isoelectric focusing performed at 20°C with a Pharmacia Multiphor II. Focusing was performed for 6 h at 100 V, 5 h at 300 V, 2 h at 600 V, 1 h at 1000 V, and 3 h at 3000 V, for a total of approximately 13600 Vh. After isoelectric focusing, the strips were stored at 80°C until required for the second dimension separation. Focused IPG strips were equilibrated in 10-ml plastic Falcon tubes with approximately 5 ml of 6 M urea, 20% glycerol, 2% tributylphosphine, 0.375% Tris and 2.5% acrylamide, pH 8.8, with gentle rocking for 20 min.
Second-dimension gels were 1.5-mm-thick prepoured 1020% Ready Gels (Bio-Rad), run using either the Mini Protean II or the Protean II Xi Multicell, both also from Bio-Rad. Either Coomasie low-molecular-weight or silver low-molecular-weight proteins (Bio-Rad) were used as molecular weight markers. Cathode running buffer was 192 mM glycine, 0.1% SDS, adjusted to pH 8.3 with Tris base. Equilibrated strips were embedded on top of the SDSPAGE gels using molten 0.5% agarose in cathode buffer, and the gels were run at a constant current of 3 mA per gel for 1 h and then 12 mA per gel for 2 h or until the dye front ran off the bottom of the gel.
Gels were stained using either silver diamine or Coomassie colloidal G-250 (stained at least for 24 h in 0.1% colloidal Coomassie in 30% methanol) stains. Silver diamine staining was carried out as follows: SDSPAGE gels were first placed in a fixative, 40% methanol, 10% acetic acid for 30 min, and then into a second fixer (45 g anhydrous sodium acetate in 30% methanol, 0.5% glutraldehyde) for 30 min. After fixing, the gels were washed for 3 x 10 min with water, then incubated 2 x 30 min with 0.05% 2,7 naphthaline-disulfonic acid, then washed 2 x 10 min with water, followed by a 30-min incubation in a solution of silver (1.5% ammonia, 0.08% NaOH, carefully mixed with 0.6% silver nitrate). The gels were then washed for 3 x 4 min with water and developed in 0.01% citric acid, 0.1% formaldehyde for 5 min. Development was stopped with 5% acetic acid for 10 min with subsequent washing 2 x 10 min with water. Stained gels were immediately imaged on a Molecular Dynamics SI densitometer before further use or analysis.
Ion chromatography
Separation of sulfate from phosphate was determined as described by Harrison and Packer (2000). Approximately 1 nmol of protein was dried on a Savant Speed-Vac and resuspended in 50 µl of 4 M HCl. Samples were then hydrolyzed in screw-capped Eppendorf tubes at 100°C for 4 h. Hydrolyzed samples were dried and twice resuspended in 50 µl of water and redried to reduce the levels of residual HCl present in the sample. Dry, hydrolyzed samples and nonhydrolyzed controls were resuspended in water and analyzed for free phosphate or sulfate by ion chromatography. Ion chromatography was performed on a Dionex HPLC using a Dionex IonPacAS11 analytical ion-exchange column (250 mm x 4 mm) with a Dionex DX500 LC pump and a postcolumn conductivity detector with an in-line AMMS anion suppressor with neutralization using 0.5 N H2SO4. Separation was achieved over a shallow, concave gradient from 5% to 30% NaOH over 15 min at a constant flow rate of 1 ml/min. The column was reequilibrated with 5% NaOH, 95% water for 20 min between analyses. Sodium phosphate, sodium sulfate, sodium chloride, and sodium acetate were used as standards.
Solid-phase Edman degradation
Identification of glycosylated amino acids and N-terminal sequencing was performed by solid-phase protein sequencing on a Hewlett-Packard protein sequenator as previously described (Gooley and Williams, 1997).
General methods
Amino acid analysis and phosphoamino acid analysis were performed by the Australian Proteome Analysis Facility. Protein concentration of the AXE stock sample was measured colorimetrically using a Bio-Rad DC Protein Assay kit according to the manufacturers instructions.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
2 To whom correspondence should be addressed
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chen, H-M., Ford, C., and Reilly, P.J. (1994) Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N- glycosylation and inactivation by deamination. Biochem. J., 301, 275281.[ISI][Medline]
Eriksen, S.H., Jenson, B., and Olsen, J. (1998) Effect of N-linked glycosylation on secretion, activity, and stability of -amylase from Aspergillus oryzae. Curr. Microbiol., 37, 117122.[CrossRef][ISI][Medline]
Gooley, A.A. and Williams, K.L. (1997) How to find, identify and quantitate the sugars on proteins. Nature, 385, 557559.[CrossRef][ISI][Medline]
Grohmann, K., Mitchell, D.J., Himmel, M.E., Dale, B.E., and Schoeder, H.A. (1989) The role of ester groups in resistance of plant cell wall polysaccharides to enzymatic hydrolysis. Appl. Biochem. Biotechnol., 20/21, 4561.
Gum, E.K. and Brown, R.D. (1976) Structural characterisation of a glycoprotein cellulase, 1, 4-ß-D-glucan cellobiohydrolase C from Trichoderma viride. Biochim. Biophys. Acta, 446, 371386.[ISI][Medline]
Gunnarsson, A., Svensson, B., Nilson, B., and Svensson, S. (1984) Structural studies on the O-glycosidically linked carbohydrate chains of glucoamylase G1 from Aspergillus niger. Eur. J. Biochem., 145, 463467.[Abstract]
Harrison, M.J. and Packer, N.H.(2000) Measurement of sulfate in mucins. Methods Mol. Biol., 125, 211216.[Medline]
Harrison, M.J., Nouwens, A.S., Jardine, D.R., Zachara, N.E., Gooley, A.A., Nevalainen, H., and Packer, N. (1998) Modified glycosylation of cellobiohydrolase I from high cellulase-producing mutant strain of Trichoderma reesei. Eur. J. Biochem., 256, 119127.[Abstract]
Haynes, P.A. (1998) Phosphoglycosylation: a new structural class of glycosylation? Glycobiology, 8, 15.
Hooper, L.V., Manzella, S.M., and Baenziger, J.U. (1996) From legumes to leukocytes: biological roles for sulfated carbohydrates. FASEB J., 10, 11371146.
Klarskov, K., Piens, K., Ståhlberg, J., Høi, P.B., van Beeumen, J., and Claeyssens, M. (1997) Cellobiohydrolase I from Trichoderma reesei: identification of an active-site nucleophile and additional information on sequence including glycosylation pattern of the core protein. Carbohydr. Res., 304, 143154.[CrossRef][ISI][Medline]
Lis, H. and Sharon, N. (1993) Protein glycosylation structural and functional aspects. Eur. J. Biochem., 218, 127.[Abstract]
Maras, M., De-Bruyn, A., Schraml, J., Herdewijn, P., Claeyssens, M., Fiers, W., and Contreras, R. (1997) Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RutC-30. Eur. J. Biochem., 245, 617625.[Abstract]
Margolles-Clark, E., Tenkanen, M., Söderlund, H., and Penttilä, M. (1996) Acetyl xylan esterase from Trichoderma reesei contains an active serine and cellulose binding domain. Eur. J. Biochem., 237, 553560.[Abstract]
Matsuo, T. and Mizuno, T. (1974) Acetyl groups in native glucomannan from ester lily bulbs. Agric. Biol. Chem., 38, 465466.[ISI]
Morris, E.J. and Bacon, J.S.D. (1977) The fate of acetyl groups and sugar components during the digestion of cell walls in sheep. J. Agric. Sci. Camb., 89, 327340.[ISI]
Neustroev, K.N., Golubev, A.M., Firsov, L.M., Ibatullin, F.M., Potaevich, I., and Makarov, A. (1993) Effect of modification of carbohydrate component on properties of glucoamylase. FEBS Lett., 2, 157160.[CrossRef]
Nevalainen, H., Harrison, M., Jardine, D., Zachara, N., Paloheimo, M., Suominen, P., Gooley, A.A., and Packer, N. (1997) Glycosylation of cellobiohydrolase I from Trichoderma reesei. In Claeyssens, M., Nerinckx, W., and Piens, K. (eds.), The carbohydrases from Trichoderma reesei and other microorganismsstructures, biochemistry, genetics and applications. Royal Society of Chemistry, UK, pp. 335344.
Packer, N.H., Lawson, M.A. Jardine, D.R., and Redmond, J.W. (1998) A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J. 15, 737747.[CrossRef][ISI][Medline]
Poutanen, K. and Puls, J. (1988) Characteristics of Trichoderma reesei ß-xylosidase and its use in the hydrolysis of solubilized xylans. Appl. Microbiol. Biotechnol., 28, 425432.[ISI]
Poutanen, K. and Sundberg, M. (1988) An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol., 28, 419424.[ISI]
Poutanen, K., Rättö, M., Puls, J. and Viikari, L. (1987) Evaluation of different microbial xylanolytic systems. J. Biotechnol., 6, 49.[ISI]
Poutanen, K., Sundberg, M., Korte, H., and Puls, J. (1990) Deacetylation of xylans by acetyl esterases of Trichoderma reesei. Appl. Microbiol. Biotechnol., 33, 506510.[ISI]
Savelev, A., Eneyskaya, E.V., Isaeva-Ivanova, L.S., Shabalin, K.A., Golubev, A., and Neustroev, K.N. (1997) The carbohydrate moiety of a-galactosidase from Trichoderma reesei. Glycoconj. J., 14, 897905.[CrossRef][ISI][Medline]
Sundberg, M. and Poutanen, K. (1991) Purification and properties of two acetyl xylan esterases of Trichoderma reesei. Biotechnol. Appl. Biochem., 13, 111.[ISI]
Takayanagi, T., Kushida K., Idonuma K., and Ajisaka K. (1992) Novel N-linked oligo-mannose type oligosaccharides containing an alpha-D-galactofuranosyl linkage found in alpha-D-galactosidase from Aspergillus niger. Glycoconj. J., 9, 229234.[ISI][Medline]
Tenkanen, M. (1998) Action of Trichoderma reesei and Aspergillus oryzae esterases in the deacetylation of hemicelluloses. Biotechnol. Appl. Biochem., 27, 1924.[ISI][Medline]
Van Tilbeurgh, H., Tomme, P., Claeyssens, M., Bhikhabhai, R., and Pettersson, G. (1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reeseiseparation of functional domains. FEBS Lett., 204, 223227.[CrossRef][ISI]
Williamson, G., Belshaw, N.J., Noel, T.R., Ring, S.G., and Williamson, P. (1992) O-glycosylation and stability. Unfolding of glucoamylase induced by heat and guanidine hydrochloride. Eur. J. Biochem., 207, 661670.[Abstract]
Wood, J.M. and McCrae, S.I. (1986) The effect of acetyl group on the hydrolysis of ryegrass cell walls by xylanase and cellulase of Trichoderma koningii. Phytochemistry, 25, 10531055.[CrossRef][ISI]
Yan, J.X., Wilkins, M.R., Ou, K., Gooley, A.A., Williams, K.W., Sanchez, J.-C., Golaz, O., Pasquali, C., and Hochstrasser, D.L. (1996) Large-scale amino-acid analysis for proteome studies. J. Chrom., 736, 291302.[CrossRef][ISI]