A xyloglucan-specific endo-[beta]-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

Markus Pauly, Lene N.Andersen1, Sakari Kauppinen1, Lene V.Kofod1, William S.York2, Peter Albersheim and Alan Darvill

Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA and 1Novo Nordisk A/S, Novo Alle, DK-2880 Bagsværd, Denmark

Received on May 4, 1998; revised on June 2, 1998; accepted on June 7, 1998

A full-length c-DNA encoding a xyloglucan-specific endo-[beta]-1,4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30°C. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo-[beta]-1,4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Km of the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 µmol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.

Key words: Aspergillus aculeatus/endoglucanase/xyloglucan/ xyloglucan-specific endo-[beta]-1,4-glucanase

Introduction

Xyloglucan (XG) is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone ([beta]-1,4-linked glucosyl residues) that is frequently substituted with side chains. The side chains are composed of xylosyl, galactosyl, fucosyl, and/or arabinosyl residues (Hayashi, 1989). XG is believed to function in the primary wall of plants by cross-linking cellulose microfibrils, forming a cellulose-XG network (Bauer et al., 1973) that is considered important for the structural integrity of the walls. Another biological function of XG is to act as a repository for XG subunit oligosaccharides that are physiologically active as regulators of plant cell growth (York et al., 1984). XG subunits may also modulate the action of a XG endotransglycosylase (XET), a cell wall-associated enzyme that may play a role in the elongation of plant cell walls (Fry et al., 1992). Therefore, XG metabolism might play an important role in wall loosening and consequent cell expansion.

The fine structural features of XG reflect its metabolic history, and so analysis of the oligosaccharide subunit composition of XGs isolated from plant tissues under varying physiological conditions can reveal a correlation between cell expansion and XG metabolism (Guillen et al., 1995). XG is often solubilized by treating cell walls with strong alkali (e.g., 4 M KOH), perhaps by disruption of the hydrogen bonds between XG and cellulose (Hayashi et al., 1987). However, this harsh chemical treatment of the wall destroys some structural features of XG, such as O-acetyl substituents, which have been found on XG isolated from various sources (York et al., 1988, 1996; Kiefer et al., 1989; Maruyama et al., 1996; Sims et al., 1996). Therefore, enzymatic solubilization of XG is preferable because it yields native xyloglucan oligosaccharides (XGOs). Our group previously used a commercially available cellulase preparation to release XGOs from pea stem cell walls (Guillen et al., 1995). However, that cellulase preparation also possessed xylan- and cellulose-degrading activities. Treatment of the cell wall with such an enzyme preparation results in a mixture of products consisting of XGOs, xylan-derived oligosaccharides, and cellulose-derived oligosaccharides. The task of purifying the enzyme-generated XGOs is simplified by avoiding solubilization of other cell wall components.

The filamentous fungus Aspergillus aculeatus produces a large variety of cellulolytic enzymes including three endoglucanases, three exoglucanases, and three [beta]-glucosidases, all of which have been previously purified and characterized (Murao et al., 1988). In addition a cDNA encoding one of the endoglucanases, FI-CMCase, has been cloned and sequenced (Ooi et al., 1990a,b). This organism also proved to be a good source of a xyloglucan-specific endo-[beta]-1,4-glucanase (XEG) that is a potentially powerful tool for the structural analysis of cell wall XG.

This report describes the isolation, expression, purification, and characterization of an endo-[beta]-1,4-glucanohydrolase (XEG) that solubilizes only XGOs from plant cell walls, thus facilitating structural analysis of the XGOs with the goal of determining their roles in plant cell development.

Results

Isolation and characterization of cDNA clones

A cDNA library from Aspergillus aculeatus was constructed in the yeast expression vector pYES 2.0 and transformed into S.cerevisiae. The transformants were screened for endoglucanase activity by replica plating the yeast colonies onto agar plates containing AZCL XG. Of the 25,000 yeast transformants screened, 71 colonies showed endoglucanase activity, of which 67 contained a 1.1 kb cDNA insert. Nucleotide sequence analysis revealed that all 67 represented transcripts of a single gene later demonstrated to encode a XG-specific endo-[beta]-1,4-glucanase (XEG). The nucleotide sequence of the XEG cDNA was submitted to the GenBank/EMBL Data Bank (accession number: AF043595). The deduced amino acid sequence of XEG is presented in Figure 1. The cDNA encodes an apparent signal sequence of 14 amino acids with a typical signal cleavage site between Ala-14 and Ala-15. The secreted XEG has a predicted molecular mass of 23,756 Da.


Figure 1. Alignment of the deduced amino acid sequence of XEG with that of FI-CMCase, both from A.aculeatus. Identical amino acids are boxed, and the predicted signal peptide is underlined. The nucleotide sequence of the XEG cDNA has accession number AF043595 in the GenBank/EMBL data bank.

A 1.0 kb cDNA insert representing transcripts of a gene identical to the FI-CMCase gene previously described by Ooi et al. (1990a,b) was present in the four other clones as determined by nucleotide sequence analysis of their 5[prime] termini. Alignment of the deduced amino acid sequence of XEG and the FI-CMCase reveals 49% identity (Figure 1). Both endoglucanases belong to family 12 of glycosyl hydrolases (Henrissat and Bairoch, 1993).

Yeast clones expressing XEG and FI-CMCase, respectively, were simultaneously tested on agar plates containing AZCL-XG, AZCL-hydroxyethylcellulose, or AZCL-[beta]-glucan. The FI-CMCase-secreting clone is active on all three substrates, while the XEG-secreting clone degrades only AZCL-XG.

Heterologous expression and purification of recombinant XEG

The XEG cDNA was subcloned into the fungal expression vector pHD464 and transformed into Aspergillus oryzae strain A1560 by cotransformation with amdS+ (Christensen et al., 1988) to obtain high level expression of XEG. The transformants were tested for endoglucanase activity, and the highest yielding transformant was grown in one-liter fermentors. The culture supernatant exhibited endoglucanase activity against polymeric tamarind XG and exoglycosidase activity against BEPS-XGOs (data not shown). This suggested that the supernatant contained endoglucanase along with small amounts of [alpha]-fucosidase, [beta]-galactosidase, [alpha]-xylosidase, and/or [beta]-glucosidase. The concentrated, dialyzed culture supernatant of the transformed A.oryzae was subjected to anion-exchange chromatography (Figure 2A). Fractions were collected and assayed for endoglucanase activity and glycosidase activity. The fractions that eluted at 5-15 min and 35-42 min exhibited glycosidase activity, but the major protein peak (44-60 min) exhibited only endoglucanase activity. SDS-PAGE analysis of this major protein peak indicated the presence of several different protein bands (data not shown). Therefore, the major protein peak was subjected to further purification by gel permeation chromatography on a Superose 12 column (Figure 2B). The resulting enzyme (termed XEG) was homogeneous as shown by SDS-PAGE (Figure 3) and MALDI-TOF MS (Figure 4).


Figure 2. Purification of the recombinant XEG from the culture supernatant of the Aspergillus oryzae transformant. (A) Anion-exchange chromatography (Econo-Q-cartridge; for details, see Materials and methods). Fractions under the bar were pooled and subjected to gel permeation chromatography. (B) Gel permeation chromatography (Superose 12; for details, see Materials and methods). Fractions under the bar were pooled and represent the purified XEG. Solid line, protein (A280); dotted line, salt gradient; multiplication signs, glycosidase activity on BEPS-XGO; solid squares, endoglucanase activity on tamarind XG.


Figure 3. SDS-PAGE of purified XEG. Lane 1, standard protein molecular weight markers; lane 2, purified XEG.

Characterization of XEG

XEG has an apparent molecular mass of 34 kDa as determined by SDS-PAGE (Figure 3). However, MS analysis reveals an average molecular mass of ~ 23.6 kDa (Figure 4), which approximates the molecular mass of 23,756 Da deduced from the amino acid sequence (Figure 1). The deduced XEG sequence does not have any consensus N-glycosylation sites (Asn-X-Ser or Asn-X-Thr), and lack of glycosylation is in agreement with the MS-data. Its measured isoelectric point of 3.4 is in reasonable agreement with the theoretical value of 3.7.

Characterization of the enzymatic properties of XEG

XEG is most stable at pH 3.0-3.8. Its stability declines sharply below pH 2.8 and above pH 5. XEG is very stable below 35°C, but at 50°C it loses 80% of its activity within 2 h. The Km was determined to be 3.6 mg/ml with a specific activity of 260 µmol/min per mg protein, corresponding to a kcat of 113 s-1 using tamarind XG as the substrate.

Glycosyl hydrolases cleave their substrate in a stereo-selective manner. The anomeric configuration of a glycosyl residue is either inverted (via a single displacement reaction) or retained (via a double displacement reaction; Sinnott, 1990) as a result of the hydrolase-catalyzed reaction. The stereochemistry of XG hydrolysis, with tamarind XG as the substrate, was observed by 1H-NMR spectroscopy (Figure 5). Sufficient amounts of XEG were added (10 units) to ensure a complete hydrolysis of the XG within ~2 min after the addition of enzyme. Immediately after digestion of the XG, the anomeric signals of newly formed reducing [beta]-glucose residues are visible at 4.65 ppm (Friebolin, 1991; York et al., 1988) in the 1H-NMR spectrum. The anomeric signals of reducing [alpha]-glucose at 5.22 ppm do not appear until 45 min after XEG digestion of the XG is complete indicating they are a result of mutarotation. Thus, XEG cleaves glycosidic bonds with retention of the [beta]-configuration of the glycosyl residues.


Figure 4. Matrix-assisted laser desorption/ionization time-of-flight MS of purified recombinant XEG. The number of charges and aggregation state of the ions are indicated. For example, [3M + 2H]2+ indicates an aggregate of three polypeptide chains and two protons with a net charge of +2 .

When examined by 1H-NMR the XGO products produced by treatment of tamarind XG with XEG (Figure 5) are indistinguishable from those generated by commercially available cellulase (data not shown). This indicates that XEG, like all other endoglucanases investigated to date from Trichoderma, generates XGOs in which the reducing glucosyl residue has no side chain attached to it (Kooiman, 1961).

XEG was tested for its ability to hydrolyze a variety of substrates obtained from plant cell walls (see Materials and methods) including XG (from tamarind and extracellular polysaccharides from the medium of cultured bean cells (BEPS-XG)), other hemicellulosic polysaccharides (arabinoxylan, (1,3),(1,4)-[beta]-d-glucan, and galactomannan), cellulosic polysaccharides (Avicel and the chemically modified substrate (CMC 7)), and pectins (polygalacturonic acid, partially methylesterified polygalacturonic acid, and arabinogalactan). Except for the XGs, XEG does not hydrolyze any plant cell wall polysaccharide tested. Interestingly, XEG is only half as active on BEPS-XG as on tamarind XG. The main difference between tamarind XG and BEPS-XG is that the [beta]-galactosyl residues in the side chains of BEPS-XG are often substituted at O-2 with terminal [alpha]-l-fucosyl residues and at O-3, O-4, and O-6 with O-acetyl substituents.


Figure 5. Anomeric region of 1H-NMR spectra of tamarind XG treated with the XEG for different times. The rapid appearance of the [beta]-anomeric proton at 4.65 ppm and subsequent appearance of the [alpha]-anomeric proton at 5.22 ppm indicate that XEG is a retaining hydrolase. Time point 0 was recorded just before the addition of the XEG.

Solubilization of XGOs by XEG

The ability of XEG to solubilize XGOs from plant cell wall tissue was compared to that of a commonly used, commercially available cellulase preparation from Trichoderma reesei as described in Material and methods. Tissue from whole etiolated pea stems was used. The partially depectinated cell wall was incubated for 24 h with 1 unit of either XEG or cellulase in the presence of thimerosal. After removing the enzymes by anion-exchange chromatography of the filtrates, the solubilized carbohydrates were desalted on a Sephadex G-10 column, which also resolved two carbohydrate-containing peaks (Figure 6). MS analysis showed that the G-10 void peak contained XGOs, whereas the partially included peak consisted solely of hexoses and hexobioses, which were likely to be glucose and cellobiose derived from cellulose. The amount of carbohydrates present in these peaks was determined by integration of the anthrone response of the fractions and by comparison to an anthrone standard curve of BEPS-XGOs (for the void peak) and glucose (for the partially included peak, Figure 6). The XEG treatment released approximately 33 mg XGOs and only very little glucose and cellobiose (1 mg). By comparison, the cellulase treatment resulted in the solubilization of only 14 mg of XGOs but 10 mg of glucose and cellobiose. Digestion of the partially depectinated cell wall with 1 unit XEG under the conditions used therefore results in a higher and purer yield of XGOs than digestion with 1 unit of the cellulase under the same conditions.


Figure 6. Treatment of pea stem tissue with XEG or cellulase. Partially depectinated pea stem cell wall (0.5 g) was treated with 1 unit of each enzyme for 24 h at 37°C. The Sephadex G-10 profiles of the filtrates (left side) indicate two carbohydrate-containing peaks, a void peak (solid squares) containing XGOs, and a partially included peak (open squares) containing glucose and cellobiose. The solubilized amounts of XGOs (black columns, right side) and glucose plus cellobiose (white columns) were calculated by integrating the carbohydrate containing peaks of the G-10 profiles and comparing them to anthrone standard curves of BEPS-XGOs and glucose. Solid triangles, conductivity (in mS) representing the ionic content of the fractions (salts); solid squares and open squares, sugar content of collected fractions using the anthrone assay (A620); solid squares, XGOs (black bar in column chart); open squares, cellobiose and glucose (white bar in column chart). Error bars indicate the standard deviation of three experiments.

However, it should be noted that if the cell wall is treated with excessive amounts of XEG or cellulase (20 units, 48 h), the cellulase treatment actually solubilizes more XGOs than XEG (data not shown). These data have implications regarding the macromolecular organization of plant cell walls and will be more thoroughly described in a forthcoming publication.

Discussion

A cDNA encoding a XG-specific endo-[beta]-1,4-glucanase (XEG) was isolated from Aspergillus aculeatus and successfully expressed heterologously. The recombinant XEG was characterized, and its enzymatic properties were studied. Amino acid sequence analysis revealed that XEG has 49% homology to a FI-CMCase from the same organism (Figure 1) and therefore belongs to the family 12 of glycosyl hydrolases (Henrissat and Bairoch, 1993).

Comparison of XEG to other family 12 glucanases

To date, nine endoglucanases (EGs) of this family have been identified; their chemical and enzymatic properties are summarized in Table I. Most of these enzymes are relatively small proteins (23-27 kDa) consisting only of a catalytic protein domain, although the Eg1S and CelB from Streptomyces contain additional cellulose-binding (CBD) and linker domains, which explains their higher molecular weight. A CBD has been shown to facilitate the hydrolysis of Avicel (microcrystalline cellulose; Tomme et al., 1988; Ahn et al., 1997).

Table I. Biochemical characteristics of family 12 endoglucanases
Enzymea Organism MW (kDa) pI Substrateb CBD Catalytic mechanism
XEG Aspergillus aculeatus 23.6c/35d 3.4 Only XG No Retention of anomeric configuration
FI-CMCase Aspergillus aculeatus 25d 4.8 XGg CMC no Avicel no nd
CMCase I Aspergillus kawachii 27d no CMC XG nd nd nd
Cel A Aspergillus oryzae KBN616 24e/31d nd CMC XG nd nd nd
EG III Humicola insolens nd nd CMC XG nd nd Retention of anomeric configuration
EG IV (III) Trichoderma viride 23.5d 7.7 XGf CMC no nd
Cel S Erwinia carotophora
ssp. Carotophora
26d 5.5 CMC xylan
no Avicel XG nd
nd nd
Eg1Sh Streptomyces rochei A2 43d nd CMC XG nd yes nd
Cel BH Streptomyces lividans 66 36d nd CMC XG nd yes nd
nd, Not determined.
aData obtained for FI-CMCase from Murao et al., 1988; CMCas I from Sakamato et al., 1995; Cel A from Kitamato et al., 1996; EG III from Schou et al., 1993; EG IV (also known as EG III; Ward et al., 1993) from Beldman et al., 1985; Cel S from Saarilahti et al., 1990; Eg1S from Perito et al., 1994; Cel B from Wittmann et al., 1994.
bXG nd, XG not tested as substrate; CBD, cellulose-binding domain.
cObtained by MS analysis.
dObtained by SDS-PAGE.
eObtained from sequence.
fAccording to Vincken et al., 1997.
gAccording to Dalboge et al., 1994.
hProbably a member of a different subfamily.

Crystallographic analysis and site-directed mutagenesis of sequence related xylanases have led to the proposal that the Glu-134 and Glu-218 of FI-CMCase are essential for catalysis (Torronnen et al., 1993). The homologous residues Glu-133 and Glu-219 of XEG are likely to play an important role in catalysis, as these residues and the motif GTEPFTG containing Glu-219 are conserved in FI-CMCase and XEG. This motif is also conserved in the other family 12 EGs including CMCaseI and Cel A from Aspergillus, EG III from Humicola, and EG III from Trichoderma (Ward et al., 1993). The motif is slightly modified in CelS by substituting an isoleucine for proline and a glycine for tyrosine (GTEIFGG) (Saarilahti et al., 1990). However, this domain is not conserved in the Streptomyces EGs, which, considering their catalytic motif, molecular weight and CBD, are probably representatives of a different subfamily.

The ability of EGs to cleave small chromophoric glycosides has led to the suggestion that their substrate specificity is conserved within each EG family (Claeyssens and Henrissat, 1992). Conservation of substrate specificity is not observed with polysaccharides, since XEG does not hydrolyze CMC, which is a substrate for all other known EGs in family 12. However, the FI-CMCase from A.aculeatus (Dalboge et al., 1994) and the EG III from T.viride (Vincken et al., 1994, 1997) have activity against XG. Therefore, the other EGs in family 12 may also have activity against XG.

It has also been proposed that the EGs within a family share the same catalytic mechanism (Claeyssens and Henrissat, 1992; Schou et al., 1993). The retention of anomeric configuration by the XEG supports this proposal.

Comparison of XEG to other XG-specific endoglucanases

To date three XG-specific endo-[beta]-1,4-glucanases are known, all from plants. One, NXG1, was purified from cotyledons of germinated nasturtium seeds (Tropaeolum majus L.; Edwards et al., 1985, 1986). It was later shown that, in addition to hydrolytic activity, NXG1 also exhibits xyloglucan endotransglycosylase (XET) activity (Fanutti et al., 1993). Subsequent cloning and sequencing of NXG1 revealed homology to several XETs from various plant species and family 16 bacterial [beta]-1,3-1,4-glucanases (Borris et al., 1990; Xu et al., 1995), but no homology to other plant EGs.

Another XG-specific EG with glycosyltransferase activity has been isolated from the cell walls of azuki bean epicotyls (Verma et al., 1975). However, it apparently hydrolyzes XG into fragments of about 50 kDa rather than XGOs, indicating a mode of catalysis different from that of XEG.

A XG-specific EG has been isolated from auxin-treated pea stems (Pisum sativum L.) (Matsumoto et al., 1997). However, the amino acid sequence of the pea EG is not known, but it is more likely related to nasturtium NXG1 than to XEG, because plant EGs are distinct from fungal and bacterial EGs (Tomme et al., 1995).

Conclusion

XEG is the first representative of a fungal enzyme exhibiting XG-specific EG activity. XEG is unique in its substrate specificity when compared to other family 12 EGs with similar amino acid sequences, yet as a fungal enzyme it is distinct from plant XG-specific EGs. Therefore, XEG should facilitate future studies aimed at determining the structural basis for XG specificity in EGs. XEG solubilizes selectively XGOs from complex polysaccharide aggregates such as plant cell walls, making it a very useful tool for XG research to provide further insights into the structure and metabolism of XG in the plant cell walls of higher plants.

Materials and methods

Materials

Tamarind XG and tamarind XGOs were prepared as described previously (York et al., 1993). XGOs from the medium of cultured cannolino bean cells (BEPS-XG; Phaeseolus vulgaris var. cannolino; origin, Italy) were prepared as described previously (Wilder and Albersheim, 1973) via ethanol precipitation and subsequent digestion with cellulase (Megazyme, Bray, Ireland). Carboxymethylcellulose (CMC) 7 was purchased from Hercules (Wilmington, DE). Azurine-dyed and cross-linked XG (AZCL-XG), AZCL hydroxyethylcellulose, AZCL [beta]-glucan, and wheat flour arabinoxylan were purchased from Megazyme. (1,3),(1,4)-[beta]-d-Glucan from barley flour was obtained from Biosupplies (Parkville, Australia), and Avicel PH101 from Fluka (Milwaukee, WI). Arabinogalactan from larchwood, gum galactomannan from locust bean, methylesterified pectin from citrus fruit, and polygalacturonic acid were all purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Sigma or Aldrich (Milwaukee, WI) unless otherwise noted.

Fungal strains and growth conditions

Aspergillus aculeatus strain KSM510 was cultured as previously described (Kofod et al., 1994); the mycelium was harvested after 5 days growth at 30°C, frozen in liquid N2, and stored at -80°C. Aspergillus oryzae A1560 (Christensen et al., 1988) transformants were grown in YP medium containing 35 g/l of maltodextrin.

Expression cloning

PolyA+ RNA was isolated from the mycelium of A.aculeatus, cDNA was synthesized and ligated into the pYES 2.0 vector, and the library transformed into E.coli as described previously (Kofod et al., 1994). The library, consisting of 1.5 × 106 clones, was stored as individual pools (5000-7000 colony-forming units/pool).

Plasmid DNA from a cDNA library pool was transformed into S.cerevisiae W3124 (van den Hazel et al., 1992) by electroporation (Becker and Gurante, 1991). The transformants were plated on SC plates (Sherman, 1991) containing 2% glucose and then incubated at 30°C for 3 days. The colonies were replicated onto a set of SC plates containing 2% galactose and either 0.1% of AZCL-XG, AZCL-hydroxyethylcellulose, or AZCL [beta]-glucan for detection of endoglucanase activity. After incubation at 30°C for 3-5 days, positive clones were identified by their ability to form blue halos only on AZCL-XG as substrate. The halos resulted from solubilization of dyed XG fragments from the insoluble substrate.

Total DNA from the positive yeast colonies was isolated as described previously (Strathern and Higgins, 1991), and the insert-containing pYES 2.0 clones were rescued by selecting ampicillin-resistant transformants of E.coli MC1061.

Nucleotide sequence analysis

The nucleotide sequence of the XEG cDNA clone was determined for both strands by the dideoxy chain termination method (Sanger et al., 1977) using fluorescence labeled terminators. Qiagen-purified plasmid DNA (Qiagen, Los Angeles, CA) was sequenced with the Taq deoxy terminal cycle sequencing kit (Perkin Elmer, Norwalk, CT) and either pYES 2.0 polylinker primers (Invitrogen, Carlsbad, CA) or synthetic oligonucleotide primers using an Applied Biosystems 373A automated sequencer according to the manufacturer's instructions. Analysis of the sequence data was performed as described previously (Devereux et al., 1984).

Expression of recombinant enzyme in A.oryzae

Plasmid DNA from the XEG clone was digested with appropriate restriction enzymes. The insert was purified by agarose gel electrophoresis and ligated into an Aspergillus expression vector, pHD464 (Dalboge and Heldt-Hansen, 1994), followed by transformation of the construct into E.coli MC 1061 (Sambrook et al., 1997). Plasmid DNA was isolated from E.coli by the Qiagen method and cotransformed into A.oryzae A1560 as described (Christensen et al., 1988). The amdS+ transformants were assayed for enzyme activity against AZCL-XG, and the best producer was used for XEG production via fermentation.

Protein determination

Protein content was determined by the Bradford method (Bio-Rad, Hercules, CA) with [gamma]-globulin as the standard. The absorbance at 280 nm was used to monitor the protein in column eluents.

Purification of recombinant XEG

Recombinant XEG was purified from 1l fermentors of the selected A.oryzae transformant. Macromolecules in the culture filtrates were concentrated with an Amicon ultrafiltration unit fitted with a 10 kDa membrane. The retentate was then lyophilized, dissolved in water (concentration approximately 10 mg/ml), and dialyzed against 50 mM sodium acetate, pH 5.0, for 2 days in 12,000-14,000 MWCO tubing (Spectrum, Houston, TX). The dialyzed filtrate was applied to an anion-exchange column (Econo-Pac high Q-cartridge from Bio-Rad) that had been equilibrated with the same buffer. The column was eluted over 40 min at 1 ml/min with a linear 0-0.5 M NaCl gradient in 50 mM sodium acetate, pH 5.0. Fractions (1 ml) were collected and assayed for exoglycosidase and endo-[beta]-1,4-glucanase activity (see below). Fractions exhibiting only endo-[beta]-1,4-glucanase activity were pooled and applied to a gel permeation chromatography column (Superose 12; Pharmacia, Piscataway, NJ) that had been equilibrated with 0.3 M NaCl in 50 mM sodium acetate pH 5.0. The proteins were eluted with the same buffer at a flow rate of 0.3 ml/min. Aliquots of fractions exhibiting endo-[beta]-1,4-glucanase activity were pooled and frozen.

SDS-PAGE and isoelectric focusing

SDS-PAGE was performed in 12.5% polyacrylamide mini-gels according to the manufacturer's instructions (Hoefer SE 200 series, Pharmacia, USA). Isoelectric focusing was carried out in precast Ampholine PAG plates, pH 3.5-9.5 (Pharmacia, Sweden), according to the manufacturer's instructions. Gels were silver-stained as described previously (Blum et al., 1987).

Mass spectrometry (MS)

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was performed with a Hewlett Packard LDI 1700XP spectrometer operated at an accelerating voltage of 30 kV and an extractor voltage of 9 kV. The source pressure was approximately 8 × 10-7 Torr. Samples (approximately 10 pmol of purified XEG) were desorbed/ionized from the probe tip with 3 ns (~10.5 µJ) nitrogen laser pulses ([lambda] = 337 nm). The matrix, sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), was applied to the probe tip as a solution in methanol/water (90:10). A MALDI-TOF spectrum of a mixture of proteins (10 pmol each of cytochrome C, myoglobin, and bovine serum albumin) was used to calibrate the instrument.

Enzyme assays

XEG and other putative glycanase-containing fractions were assayed for activity against various substrates by the p-hydroxybenzoic acid hydrazide (PAHBAH) assay for reducing sugars (Lever, 1972). Tamarind XGOs were used as standards.

Endo-[beta]-1,4-glucanase activity. The assay mixture contained 500 µl of 0.1% polymeric tamarind XG in 50 mM sodium acetate, pH 5.0, and 2 µl of the sample to be assayed. The assays were incubated at room temperature (~21°C for 1 h). The enzymatic activity was determined by monitoring the generation of reducing sugar residues by the PAHBAH assay. One unit of endoglucanase activity was defined as the generation of 1 µmol of XGO per min under these conditions.

Exoglycosidase activity. The reaction consisted of 500 µl of 0.005% BEPS-XGOs in 50 mM sodium acetate, pH 5.0, and 2 µl of the sample to be assayed. The assays were incubated for 1 h at room temperature. The activity was determined by monitoring the release of reducing monosaccharides using the PAHBAH assay.

Determination of substrate specificity. XEG was tested for its ability to hydrolyze a variety of substrates. The reactions, which consisted of 500 µl of 0.1% substrate in 50 mM sodium acetate, pH 5.0, and 5 units of XEG (~20 ng), were incubated at 37°C for 30 min. The activity was determined using the PAHBAH assay to measure the amount of reducing sugar generated.

Determination of kinetic constants

Kinetic constants were determined using tamarind XG as a substrate and utilizing the PAHBAH assay for reducing sugars. Initial activities were determined with substrate concentrations of 2-30 mg/ml. Specific activity and Km were calculated from the double reciprocal plot of initial reaction rates at several substrate concentrations.

Effect of pH and temperature on activity and stability of the enzyme

The effect of pH and temperature on the activity of XEG was determined. Tamarind XG (0.5 ml of a 0.1% solution) was used as the substrate. After incubation the generation of reducing oligosaccharides was quantitated using the PAHBAH assay.

The pH stability of XEG was determined by adding 10 units of XEG to 10 µl of buffer solutions of various pH (2-9) without substrate. After 2 and 20 h of incubation at room temperature, 5 µl of these XEG solutions was added to the tamarind XG substrate solution (0.5 ml) in 50 mM sodium acetate, pH 4.5, and incubated for 30 min at 37°C. The activity was then determined as described.

For measurement of temperature stability, 5 units of XEG were incubated without substrates at various temperatures between 10°C and 80°C for 2 h, cooled to ambient temperature, added to the tamarind XG substrate solutions, and incubated for 30 min at 37°C. The activity was determined as described.

Determination of the catalytic mechanism of XEG

The catalytic mechanism of XEG was determined using NMR-spectroscopy. A reaction mixture containing 99.9% deuterium-exchanged tamarind XG (5 mg) and 50 mM sodium acetate, pH 4.5, in D2O was prepared (total volume 1 ml) in an NMR tube. A 1H-NMR spectrum of the reaction was recorded using a Bruker AMX600 spectrometer at 600 MHz and 298 K. Then 20 µl of an XEG or cellulase (Megazyme) solution (20 units, in D2O [99.9%]) was added, and spectra were recorded after 5 and 90 min.

Solubilization of XGOs from pea (Pisum sativum) plant cell walls

Tissue from the stems of 9-day-old etiolated pea plants (Pisum sativum cv. Alaska) was harvested and subjected to various treatments to obtain partially depectinated pea stem cell wall as described previously (Guillen et al., 1995). The dried, partially depectinated cell wall (0.5 g) was treated for 24 h at 37°C with 1 unit of XEG or 1 unit of a commercially available cellulase from Trichoderma reesei (Megazyme) in 50 mM sodium acetate, pH 4.5, containing 0.01% thimerosal as an antibiotic. After the reaction, the suspension was filtered through a coarse, sintered glass filter. XEG was removed from the filtrate by adjusting the pH of the extract to pH 7 with 1 M ammonium hydroxide and loading it onto a Q-Sepharose column (3 × 30 cm), equilibrated with 10 mM imidazole/HCl, pH 7.0, prior to loading. The loaded column was washed with two volumes of 10 mM imidazole buffer, and the eluant containing the solubilized carbohydrates was collected, concentrated, and desalted on a Sephadex G-10 column (2 × 85 cm, Pharmacia). Fractions were assayed for total hexose content by the anthrone assay and for salt content by measuring the conductivity (Conductometer model 1052A, Amber Science, Eugene, OR).

Acknowledgments

This research was supported by United States Department of Energy (DOE) grant DE-FG02-96ER20220 and by the DOE-funded (DE-FG05-93ER20097) Center for Plant and Microbial Complex Carbohydrates.

Abbreviations

AZCL, azurine-dyed and cross-linked; BEPS-XG, xyloglucan obtained from the medium of cultured bean cells; CBD, cellulose-binding domain; CMC, carboxymethylcellulose; EG, endo-[beta]-1,4-glucanase; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; PAHBAH, p-hydroxybenzoic acid hydrazide; PNB, p-nitrobenzylhydroxylamine; SC, sugar corn meal; XEG, xyloglucan-specific endo-[beta]-1,4-glucanase; XET, xyloglucan endotransglycosylase; XG, xyloglucan; XGO, xyloglucan oligosaccharide; YP, yeast peptone.

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