Glycobiology vol. 9 no. 1 pp. 93–100, 1999

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

Markus Pauly, Lene N.Andersen1, Sakari Kauppinen1, may also modulate the action of a XG endotransglycosylase Lene V.Kofod1, William S.York2, Peter Albersheim and (XET), a cell wall–associated enzyme that may play a role in the Alan Darvill elongation of plant cell walls (Fry et al., 1992). Therefore, XG Complex Carbohydrate Research Center and Department of Biochemistry and metabolism might play an important role in wall loosening and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, consequent cell expansion. GA 30602–4712, USA and 1Novo Nordisk A/S, Novo Alle, The fine structural features of XG reflect its metabolic history, DK-2880 Bagsværd, Denmark and so analysis of the oligosaccharide subunit composition of XGs Received on May 4, 1998; revised on June 2, 1998; accepted on June 7, 1998 isolated from plant tissues under varying physiological conditions can reveal a correlation between cell expansion and XG metabolism 2To whom correspondence should be addressed (Guillen et al., 1995). XG is often solubilized by treating cell walls A full-length c-DNA encoding a xyloglucan-specific with strong alkali (e.g., 4 M KOH), perhaps by disruption of the endo-β-1,4-glucanase (XEG) has been isolated from the hydrogen bonds between XG and (Hayashi et al., 1987). filamentous fungus Aspergillus aculeatus by expression However, this harsh chemical treatment of the wall destroys some cloning in yeast. The colonies expressing functional XEG structural features of XG, such as O-acetyl substituents, which have were identified on agar plates containing azurine-dyed cross- been found on XG isolated from various sources (York et al., 1988, linked xyloglucan. The cDNA encoding XEG was isolated, 1996; Kiefer et al., 1989; Maruyama et al., 1996; Sims et al., 1996). sequenced, cloned into an Aspergillus expression vector, and Therefore, enzymatic solubilization of XG is preferable because it transformed into Aspergillus oryzae for heterologous expression. yields native xyloglucan oligosaccharides (XGOs). Our group The recombinant enzyme was purified to apparent homo- previously used a commercially available preparation to geneity by anion-exchange and gel permeation chromato- release XGOs from pea stem cell walls (Guillen et al., 1995). graphy. The recombinant XEG has a molecular mass of However, that cellulase preparation also possessed xylan- and 23,600, an isoelectric point of 3.4, and is optimally stable at a cellulose-degrading activities. Treatment of the cell wall with such pH of 3.4 and temperature below 30_C. The enzyme hydrolyzes an enzyme preparation results in a mixture of products consisting of structurally diverse xyloglucans from various sources, but XGOs, xylan-derived oligosaccharides, and cellulose-derived oligo- hydrolyzes no other cell wall component and can therefore be saccharides. The task of purifying the enzyme-generated XGOs is considered a xyloglucan-specific endo-β-1,4-glucanohydrolase. simplified by avoiding solubilization of other cell wall components. XEG hydrolyzes its substrates with retention of the anomeric The filamentous fungus Aspergillus aculeatus produces a large variety of cellulolytic including three endoglucanases, configuration. The Km of the recombinant enzyme is 3.6 mg/ β ml, and its specific activity is 260 µmol/min per mg protein. three exoglucanases, and three -glucosidases, all of which have The enzyme was tested for its ability to solubilize xyloglucan been previously purified and characterized (Murao et al., 1988). oligosaccharides from plant cell walls. It was shown that In addition a cDNA encoding one of the endoglucanases, treatment of plant cell walls with XEG yields only xyloglucan FI-CMCase, has been cloned and sequenced (Ooi et al., 1990a,b). This organism also proved to be a good source of a xyloglucan- oligosaccharides, indicating that this enzyme can be a powerful β tool in the structural elucidation of xyloglucans. specific endo- -1,4-glucanase (XEG) that is a potentially powerful tool for the structural analysis of cell wall XG. Key words: Aspergillus aculeatus/endoglucanase/xyloglucan/ This report describes the isolation, expression, purification, xyloglucan-specific endo-β-1,4-glucanase and characterization of an endo-β-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. Introduction Xyloglucan (XG) is a major structural polysaccharide of the Results primary (growing) cell wall of higher plants. It consists of a Isolation and characterization of cDNA clones cellulosic backbone (β-1,4-linked glucosyl residues) that is frequently substituted with side chains. The side chains are A cDNA library from Aspergillus aculeatus was constructed in composed of xylosyl, galactosyl, fucosyl, and/or arabinosyl the yeast expression vector pYES 2.0 and transformed into residues (Hayashi, 1989). XG is believed to function in the S.cerevisiae. The transformants were screened for endoglucanase primary wall of plants by cross-linking cellulose microfibrils, activity by replica plating the yeast colonies onto agar plates forming a cellulose-XG network (Bauer et al., 1973) that is containing AZCL XG. Of the 25,000 yeast transformants considered important for the structural integrity of the walls. screened, 71 colonies showed endoglucanase activity, of which Another biological function of XG is to act as a repository for XG 67 contained a 1.1 kb cDNA insert. Nucleotide sequence analysis subunit oligosaccharides that are physiologically active as revealed that all 67 represented transcripts of a single gene later regulators of plant cell growth (York et al., 1984). XG subunits demonstrated to encode a XG-specific endo-β-1,4-glucanase

 1999 Oxford University Press 93 M.Pauly et al.

Fig. 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.

(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. 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′ termini. Alignment of the deduced amino acid sequence of XEG and the FI-CMCase reveals Fig. 2. Purification of the recombinant XEG from the culture supernatant of 49% identity (Figure 1). Both endoglucanases belong to family 12 the Aspergillus oryzae transformant. (A) Anion-exchange chromatography of glycosyl hydrolases (Henrissat and Bairoch, 1993). (Econo-Q-cartridge; for details, see Materials and methods). Fractions under Yeast clones expressing XEG and FI-CMCase, respectively, the bar were pooled and subjected to gel permeation chromatography. were simultaneously tested on agar plates containing AZCL-XG, (B) Gel permeation chromatography (Superose 12; for details, see Materials AZCL-hydroxyethylcellulose, or AZCL-β-glucan. The FI- and methods). Fractions under the bar were pooled and represent the purified XEG. Solid line, protein (A280); dotted line, salt gradient; multiplication CMCase-secreting clone is active on all three substrates, while the signs, glycosidase activity on BEPS-XGO; solid squares, endoglucanase XEG-secreting clone degrades only AZCL-XG. activity on tamarind XG.

Heterologous expression and purification of recombinant XEG peak (44–60 min) exhibited only endoglucanase activity. SDS– The XEG cDNA was subcloned into the fungal expression vector PAGE analysis of this major protein peak indicated the presence pHD464 and transformed into Aspergillus oryzae strain A1560 of several different protein bands (data not shown). Therefore, the by cotransformation with amdS+ (Christensen et al., 1988) to major protein peak was subjected to further purification by gel obtain high level expression of XEG. The transformants were permeation chromatography on a Superose 12 column (Figure 2B). tested for endoglucanase activity, and the highest yielding The resulting enzyme (termed XEG) was homogeneous as shown transformant was grown in one-liter fermentors. The culture by SDS–PAGE (Figure 3) and MALDI-TOF MS (Figure 4). supernatant exhibited endoglucanase activity against polymeric tamarind XG and exoglycosidase activity against BEPS-XGOs (data Characterization of XEG not shown). This suggested that the supernatant contained endo- glucanase along with small amounts of α-fucosidase, β-galacto- XEG has an apparent molecular mass of 34 kDa as determined by sidase, α-xylosidase, and/or β-glucosidase. The concentrated, SDS–PAGE (Figure 3). However, MS analysis reveals an average dialyzed culture supernatant of the transformed A.oryzae was molecular mass of ∼ 23.6 kDa (Figure 4), which approximates the subjected to anion-exchange chromatography (Figure 2A). molecular mass of 23,756 Da deduced from the amino acid Fractions were collected and assayed for endoglucanase activity sequence (Figure 1). The deduced XEG sequence does not have and glycosidase activity. The fractions that eluted at 5–15 min and any consensus N-glycosylation sites (Asn-X-Ser or Asn-X-Thr), 35–42 min exhibited glycosidase activity, but the major protein and lack of glycosylation is in agreement with the MS-data. Its

94 Xyloglucan-specific endo-β-1,4-glucanase from A.aculeatus

Fig. 3. SDS–PAGE of purified XEG. Lane 1, standard protein molecular weight markers; lane 2, purified XEG. measured isoelectric point of 3.4 is in reasonable agreement with the theoretical value of 3.7. Fig. 4. Matrix-assisted laser desorption/ionization time-of-flight MS of purified recombinant XEG. The number of charges and aggregation state of Characterization of the enzymatic properties of XEG 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 . 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 (polygalacturonic acid, partially methylesterified polygalacturonic determined to be 3.6 mg/ml with a specific activity of 260 µmol/min acid, and arabinogalactan). Except for the XGs, XEG does not –1 per mg protein, corresponding to a kcat of 113 s using tamarind hydrolyze any plant cell wall polysaccharide tested. Interestingly, XG as the substrate. XEG is only half as active on BEPS-XG as on tamarind XG. The Glycosyl hydrolases cleave their substrate in a stereo-selective main difference between tamarind XG and BEPS-XG is that the manner. The anomeric configuration of a glycosyl residue is β-galactosyl residues in the side chains of BEPS-XG are often either inverted (via a single displacement reaction) or retained substituted at O-2 with terminal α-L-fucosyl residues and at O-3, (via a double displacement reaction; Sinnott, 1990) as a result of O-4, and O-6 with O-acetyl substituents. the hydrolase-catalyzed reaction. The stereochemistry of XG hydrolysis, with tamarind XG as the substrate, was observed by Solubilization of XGOs by XEG 1H-NMR spectroscopy (Figure 5). Sufficient amounts of XEG were added (10 units) to ensure a complete hydrolysis of the XG The ability of XEG to solubilize XGOs from plant cell wall tissue within ∼2 min after the addition of enzyme. Immediately after was compared to that of a commonly used, commercially digestion of the XG, the anomeric signals of newly formed available cellulase preparation from Trichoderma reesei as reducing β- residues are visible at 4.65 ppm (Friebolin, described in Material and methods. Tissue from whole etiolated 1991; York et al., 1988) in the 1H-NMR spectrum. The anomeric pea stems was used. The partially depectinated cell wall was signals of reducing α-glucose at 5.22 ppm do not appear until 45 min incubated for 24 h with 1 unit of either XEG or cellulase in the after XEG digestion of the XG is complete indicating they are a presence of thimerosal. After removing the enzymes by anion- result of mutarotation. Thus, XEG cleaves glycosidic bonds with exchange chromatography of the filtrates, the solubilized retention of the β-configuration of the glycosyl residues. carbohydrates were desalted on a Sephadex G-10 column, which When examined by 1H-NMR the XGO products produced by also resolved two carbohydrate-containing peaks (Figure 6). MS treatment of tamarind XG with XEG (Figure 5) are indistinguishable analysis showed that the G-10 void peak contained XGOs, from those generated by commercially available cellulase (data whereas the partially included peak consisted solely of hexoses not shown). This indicates that XEG, like all other endoglucanases and hexobioses, which were likely to be glucose and cellobiose investigated to date from Trichoderma, generates XGOs in which derived from cellulose. The amount of carbohydrates present in the reducing glucosyl residue has no side chain attached to it these peaks was determined by integration of the anthrone (Kooiman, 1961). response of the fractions and by comparison to an anthrone XEG was tested for its ability to hydrolyze a variety of standard curve of BEPS-XGOs (for the void peak) and glucose substrates obtained from plant cell walls (see Materials and (for the partially included peak, Figure 6). The XEG treatment methods) including XG (from tamarind and extracellular poly- released approximately 33 mg XGOs and only very little glucose saccharides from the medium of cultured bean cells (BEPS-XG)), and cellobiose (1 mg). By comparison, the cellulase treatment other hemicellulosic polysaccharides (arabinoxylan, (1,3),(1,4)- resulted in the solubilization of only 14 mg of XGOs but 10 mg β-D-glucan, and galactomannan), cellulosic polysaccharides (Avicel of glucose and cellobiose. Digestion of the partially depectinated and the chemically modified substrate (CMC 7)), and pectins cell wall with 1 unit XEG under the conditions used therefore

95 M.Pauly et al.

Fig. 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) Fig. 5. Anomeric region of 1H-NMR spectra of tamarind XG treated with containing XGOs, and a partially included peak (open squares) containing the XEG for different times. The rapid appearance of the β-anomeric proton glucose and cellobiose. The solubilized amounts of XGOs (black columns, at 4.65 ppm and subsequent appearance of the α-anomeric proton at right side) and glucose plus cellobiose (white columns) were calculated by 5.22 ppm indicate that XEG is a retaining hydrolase. Time point 0 was integrating the carbohydrate containing peaks of the G-10 profiles and recorded just before the addition of the XEG. 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. results in a higher and purer yield of XGOs than digestion with 1 unit of the cellulase under the same conditions. However, it should be noted that if the cell wall is treated with excessive amounts of XEG or cellulase (20 units, 48 h), the from the same organism (Figure 1) and therefore belongs to the cellulase treatment actually solubilizes more XGOs than XEG family 12 of glycosyl hydrolases (Henrissat and Bairoch, 1993). (data not shown). These data have implications regarding the macromolecular organization of plant cell walls and will be more Comparison of XEG to other family 12 glucanases thoroughly described in a forthcoming publication. To date, nine endoglucanases (EGs) of this family have been identified; their chemical and enzymatic properties are summarized Discussion in Table I. Most of these enzymes are relatively small proteins (23–27 kDa) consisting only of a catalytic protein domain, A cDNA encoding a XG-specific endo-β-1,4-glucanase (XEG) although the Eg1S and CelB from Streptomyces contain additional was isolated from Aspergillus aculeatus and successfully ex- cellulose-binding (CBD) and linker domains, which explains pressed heterologously. The recombinant XEG was characterized, their higher molecular weight. A CBD has been shown to and its enzymatic properties were studied. Amino acid sequence facilitate the hydrolysis of Avicel (microcrystalline cellulose; analysis revealed that XEG has 49% homology to a FI-CMCase Tomme et al., 1988; Ahn et al., 1997).

96 Xyloglucan-specific endo-β-1,4-glucanase from A.aculeatus

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 nd 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 26d 5.5 CMC xylan nd nd ssp. Carotophora no Avicel XG 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 (XET) activity (Fanutti et al., 1993). Subsequent cloning and sequence related xylanases have led to the proposal that the sequencing of NXG1 revealed homology to several XETs from Glu-134 and Glu-218 of FI-CMCase are essential for catalysis various plant species and family 16 bacterial β-1,3–1,4-glucanases (Torronnen et al., 1993). The homologous residues Glu-133 and (Borris et al., 1990; Xu et al., 1995), but no homology to other Glu-219 of XEG are likely to play an important role in catalysis, plant EGs. as these residues and the motif GTEPFTG containing Glu-219 are Another XG-specific EG with glycosyltransferase activity has conserved in FI-CMCase and XEG. This motif is also conserved been isolated from the cell walls of azuki bean epicotyls (Verma in the other family 12 EGs including CMCaseI and Cel A from et al., 1975). However, it apparently hydrolyzes XG into fragments Aspergillus, EG III from Humicola, and EG III from Trichoderma of about 50 kDa rather than XGOs, indicating a mode of catalysis (Ward et al., 1993). The motif is slightly modified in CelS by different from that of XEG. substituting an isoleucine for proline and a glycine for tyrosine A XG-specific EG has been isolated from auxin-treated pea (GTEIFGG) (Saarilahti et al., 1990). However, this domain is not stems (Pisum sativum L.) (Matsumoto et al., 1997). However, the conserved in the Streptomyces EGs, which, considering their amino acid sequence of the pea EG is not known, but it is more likely catalytic motif, molecular weight and CBD, are probably related to nasturtium NXG1 than to XEG, because plant EGs are representatives of a different subfamily. distinct from fungal and bacterial EGs (Tomme et al., 1995). The ability of EGs to cleave small chromophoric glycosides has led to the suggestion that their substrate specificity is Conclusion conserved within each EG family (Claeyssens and Henrissat, XEG is the first representative of a fungal enzyme exhibiting 1992). Conservation of substrate specificity is not observed with XG-specific EG activity. XEG is unique in its substrate specificity polysaccharides, since XEG does not hydrolyze CMC, which is when compared to other family 12 EGs with similar amino acid a substrate for all other known EGs in family 12. However, the sequences, yet as a fungal enzyme it is distinct from plant FI-CMCase from A.aculeatus (Dalboge et al., 1994) and the EG XG-specific EGs. Therefore, XEG should facilitate future studies III from T.viride (Vincken et al., 1994, 1997) have activity against aimed at determining the structural basis for XG specificity in EGs. XG. Therefore, the other EGs in family 12 may also have activity XEG solubilizes selectively XGOs from complex polysaccharide against XG. aggregates such as plant cell walls, making it a very useful tool It has also been proposed that the EGs within a family share for XG research to provide further insights into the structure and the same catalytic mechanism (Claeyssens and Henrissat, 1992; metabolism of XG in the plant cell walls of higher plants. Schou et al., 1993). The retention of anomeric configuration by the XEG supports this proposal. Materials and methods Comparison of XEG to other XG-specific endoglucanases Materials To date three XG-specific endo-β-1,4-glucanases are known, all Tamarind XG and tamarind XGOs were prepared as described from plants. One, NXG1, was purified from cotyledons of previously (York et al., 1993). XGOs from the medium of germinated nasturtium seeds (Tropaeolum majus L.; Edwards et cultured cannolino bean cells (BEPS-XG; Phaeseolus vulgaris al., 1985, 1986). It was later shown that, in addition to hydrolytic var. cannolino; origin, Italy) were prepared as described previous- activity, NXG1 also exhibits xyloglucan endotransglycosylase ly (Wilder and Albersheim, 1973) via ethanol precipitation and

97 M.Pauly et al. subsequent digestion with cellulase (Megazyme, Bray, Ireland). electrophoresis and ligated into an Aspergillus expression vector, Carboxymethylcellulose (CMC) 7 was purchased from Hercules pHD464 (Dalboge and Heldt-Hansen, 1994), followed by (Wilmington, DE). Azurine-dyed and cross-linked XG (AZCL- transformation of the construct into E.coli MC 1061 (Sambrook XG), AZCL hydroxyethylcellulose, AZCL β-glucan, and wheat et al., 1997). Plasmid DNA was isolated from E.coli by the flour arabinoxylan were purchased from Megazyme. Qiagen method and cotransformed into A.oryzae A1560 as (1,3),(1,4)-β-D-Glucan from barley flour was obtained from described (Christensen et al., 1988). The amdS+ transformants Biosupplies (Parkville, Australia), and Avicel PH101 from Fluka were assayed for enzyme activity against AZCL-XG, and the best (Milwaukee, WI). Arabinogalactan from larchwood, gum galacto- producer was used for XEG production via fermentation. mannan from locust bean, methylesterified pectin from citrus fruit, and polygalacturonic acid were all purchased from Sigma Protein determination (St. Louis, MO). All other chemicals were purchased from Sigma Protein content was determined by the Bradford method (Bio-Rad, or Aldrich (Milwaukee, WI) unless otherwise noted. Hercules, CA) with γ-globulin as the standard. The absorbance at 280 nm was used to monitor the protein in column eluents. Fungal strains and growth conditions Aspergillus aculeatus strain KSM510 was cultured as previously Purification of recombinant XEG described (Kofod et al., 1994); the mycelium was harvested after Recombinant XEG was purified from 1l fermentors of the 5 days growth at 30_C, frozen in liquid N2, and stored at -80_C. selected A.oryzae transformant. Macromolecules in the culture Aspergillus oryzae A1560 (Christensen et al., 1988) transformants filtrates were concentrated with an Amicon ultrafiltration unit fitted were grown in YP medium containing 35 g/l of maltodextrin. with a 10 kDa membrane. The retentate was then lyophilized, dissolved in water (concentration approximately 10 mg/ml), and Expression cloning dialyzed against 50 mM sodium acetate, pH 5.0, for 2 days in 12,000–14,000 MWCO tubing (Spectrum, Houston, TX). The PolyA+ RNA was isolated from the mycelium of A.aculeatus, dialyzed filtrate was applied to an anion-exchange column cDNA was synthesized and ligated into the pYES 2.0 vector, and (Econo-Pac high Q-cartridge from Bio-Rad) that had been the library transformed into E.coli as described previously (Kofod equilibrated with the same buffer. The column was eluted over 6 et al., 1994). The library, consisting of 1.5 × 10 clones, was 40 min at 1 ml/min with a linear 0–0.5 M NaCl gradient in 50 mM stored as individual pools (5000–7000 colony-forming units/ sodium acetate, pH 5.0. Fractions (1 ml) were collected and pool). assayed for exoglycosidase and endo-β-1,4-glucanase activity Plasmid DNA from a cDNA library pool was transformed into (see below). Fractions exhibiting only endo-β-1,4-glucanase activity S.cerevisiae W3124 (van den Hazel et al., 1992) by electroporation were pooled and applied to a gel permeation chromatography (Becker and Gurante, 1991). The transformants were plated on column (Superose 12; Pharmacia, Piscataway, NJ) that had been SC plates (Sherman, 1991) containing 2% glucose and then equilibrated with 0.3 M NaCl in 50 mM sodium acetate pH 5.0. incubated at 30_C for 3 days. The colonies were replicated onto The proteins were eluted with the same buffer at a flow rate of a set of SC plates containing 2% galactose and either 0.1% of 0.3 ml/min. Aliquots of fractions exhibiting endo-β-1,4-glucanase AZCL-XG, AZCL-hydroxyethylcellulose, or AZCL β-glucan activity were pooled and frozen. for detection of endoglucanase activity. After incubation at 30_C for 3–5 days, positive clones were identified by their ability to SDS–PAGE and isoelectric focusing form blue halos only on AZCL-XG as substrate. The halos resulted from solubilization of dyed XG fragments from the SDS–PAGE was performed in 12.5% polyacrylamide mini-gels insoluble substrate. according to the manufacturer’s instructions (Hoefer SE 200 series, Total DNA from the positive yeast colonies was isolated as Pharmacia, USA). Isoelectric focusing was carried out in precast described previously (Strathern and Higgins, 1991), and the Ampholine PAG plates, pH 3.5–9.5 (Pharmacia, Sweden), insert-containing pYES 2.0 clones were rescued by selecting according to the manufacturer’s instructions. Gels were silver- ampicillin-resistant transformants of E.coli MC1061. stained as described previously (Blum et al., 1987). Mass spectrometry (MS) Nucleotide sequence analysis Matrix-assisted laser desorption/ionization time-of-flight The nucleotide sequence of the XEG cDNA clone was determined (MALDI-TOF) MS was performed with a Hewlett Packard LDI for both strands by the dideoxy chain termination method 1700XP spectrometer operated at an accelerating voltage of 30 kV (Sanger et al., 1977) using fluorescence labeled terminators. and an extractor voltage of 9 kV. The source pressure was Qiagen-purified plasmid DNA (Qiagen, Los Angeles, CA) was approximately 8 × 10–7 Torr. Samples (approximately 10 pmol of sequenced with the Taq deoxy terminal cycle sequencing kit purified XEG) were desorbed/ionized from the probe tip with 3 ns (Perkin Elmer, Norwalk, CT) and either pYES 2.0 polylinker (∼10.5 µJ) nitrogen laser pulses (λ = 337 nm). The matrix, primers (Invitrogen, Carlsbad, CA) or synthetic oligonucleotide sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), was primers using an Applied Biosystems 373A automated sequencer applied to the probe tip as a solution in methanol/water (90:10). according to the manufacturer’s instructions. Analysis of the A MALDI-TOF spectrum of a mixture of proteins (10 pmol each sequence data was performed as described previously (Devereux of cytochrome C, myoglobin, and bovine serum albumin) was et al., 1984). used to calibrate the instrument.

Expression of recombinant enzyme in A.oryzae Enzyme assays Plasmid DNA from the XEG clone was digested with appropriate XEG and other putative glycanase-containing fractions were restriction enzymes. The insert was purified by agarose gel assayed for activity against various substrates by the p-hydroxy-

98 Xyloglucan-specific endo-β-1,4-glucanase from A.aculeatus benzoic acid hydrazide (PAHBAH) assay for reducing sugars [99.9%]) was added, and spectra were recorded after 5 and (Lever, 1972). Tamarind XGOs were used as standards. 90 min.

Endo-β-1,4-glucanase activity. The assay mixture contained Solubilization of XGOs from pea (Pisum sativum) plant cell 500 µl of 0.1% polymeric tamarind XG in 50 mM sodium acetate, walls µ pH 5.0, and 2 l of the sample to be assayed. The assays were Tissue from the stems of 9-day-old etiolated pea plants (Pisum ∼ _ incubated at room temperature ( 21 C for 1 h). The enzymatic sativum cv. Alaska) was harvested and subjected to various activity was determined by monitoring the generation of reducing treatments to obtain partially depectinated pea stem cell wall as sugar residues by the PAHBAH assay. One unit of endoglucanase described previously (Guillen et al., 1995). The dried, partially µ activity was defined as the generation of 1 mol of XGO per min depectinated cell wall (0.5 g) was treated for 24 h at 37_C with under these conditions. 1 unit of XEG or 1 unit of a commercially available cellulase from Trichoderma reesei (Megazyme) in 50 mM sodium acetate, Exoglycosidase activity. The reaction consisted of 500 µl of pH 4.5, containing 0.01% thimerosal as an antibiotic. After the 0.005% BEPS-XGOs in 50 mM sodium acetate, pH 5.0, and 2 µl reaction, the suspension was filtered through a coarse, sintered of the sample to be assayed. The assays were incubated for 1 h at glass filter. XEG was removed from the filtrate by adjusting the room temperature. The activity was determined by monitoring the pH of the extract to pH 7 with 1 M ammonium hydroxide and release of reducing monosaccharides using the PAHBAH assay. loading it onto a Q-Sepharose column (3 × 30 cm), equilibrated with 10 mM imidazole/HCl, pH 7.0, prior to loading. The loaded Determination of substrate specificity. XEG was tested for its column was washed with two volumes of 10 mM imidazole ability to hydrolyze a variety of substrates. The reactions, which buffer, and the eluant containing the solubilized carbohydrates consisted of 500 µl of 0.1% substrate in 50 mM sodium acetate, was collected, concentrated, and desalted on a Sephadex G-10 pH 5.0, and 5 units of XEG (∼20 ng), were incubated at 37_C for column (2 × 85 cm, Pharmacia). Fractions were assayed for total 30 min. The activity was determined using the PAHBAH assay hexose content by the anthrone assay and for salt content by to measure the amount of reducing sugar generated. measuring the conductivity (Conductometer model 1052A, Amber Science, Eugene, OR). Determination of kinetic constants Acknowledgments Kinetic constants were determined using tamarind XG as a substrate and utilizing the PAHBAH assay for reducing sugars. This research was supported by United States Department of Initial activities were determined with substrate concentrations of Energy (DOE) grant DE-FG02–96ER20220 and by the DOE- funded (DE-FG05–93ER20097) Center for Plant and Microbial 2–30 mg/ml. Specific activity and Km were calculated from the double reciprocal plot of initial reaction rates at several substrate Complex Carbohydrates. concentrations. Abbreviations Effect of pH and temperature on activity and stability of the AZCL, azurine-dyed and cross-linked; BEPS-XG, xyloglucan enzyme obtained from the medium of cultured bean cells; CBD, cellulose-binding domain; CMC, carboxymethylcellulose; EG, The effect of pH and temperature on the activity of XEG was β determined. Tamarind XG (0.5 ml of a 0.1% solution) was used endo- -1,4-glucanase; MALDI-TOF, matrix-assisted laser as the substrate. After incubation the generation of reducing desorption/ionization time-of-flight; MS, mass spectrometry; oligosaccharides was quantitated using the PAHBAH assay. PAHBAH, p-hydroxybenzoic acid hydrazide; PNB, p-nitrobenzyl- hydroxylamine; SC, sugar corn meal; XEG, xyloglucan-specific The pH stability of XEG was determined by adding 10 units β of XEG to 10 µl of buffer solutions of various pH (2–9) without endo- -1,4-glucanase; XET, xyloglucan endotransglycosylase; XG, substrate. After 2 and 20 h of incubation at room temperature, 5 µl xyloglucan; XGO, xyloglucan oligosaccharide; YP, yeast peptone. 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 References for 30 min at 37_C. The activity was then determined as Ahn,D.H., Kim,H. and Pack,M.Y. (1997) Immobilization of β-glucosidase using described. the cellulose-binding domain of Bacillus subtilis endo-β-1,4-glucanase. For measurement of temperature stability, 5 units of XEG Biotechnol. Lett., 19, 483–486. were incubated without substrates at various temperatures Bauer,W.D., Talmadge,K.W., Keegstra,K. and Albersheim,P. (1973) The structure between 10_C and 80_C for 2 h, cooled to ambient temperature, of plant cell walls II. The hemicellulose of the walls of suspension-cultured sycamore cells. Plant Physiol., 51, 174–187. added to the tamarind XG substrate solutions, and incubated for Becker,D.M. and Gurante,L. (1991) High-efficiency transformation of yeast by 30 min at 37_C. The activity was determined as described. electroporation. Methods Enzymol., 194, 182–187. Beldman,G., Searle-van Leeuwen,M.F., Rombouts,F.M. and Voragen,F.G.J. (1985) The cellulase of Trichoderma viride. Purification, characterization and Determination of the catalytic mechanism of XEG comparison of all detectable endoglucanases, exoglucanases and β-glucosidases. Eur. J. Biochem., 146, 301–308. The catalytic mechanism of XEG was determined using NMR- Blum,H., Beier,H. and Gross,H.J. (1987) Improved silver staining of plant spectroscopy. A reaction mixture containing 99.9% deuterium- proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93–99. exchanged tamarind XG (5 mg) and 50 mM sodium acetate, Borris,R., Buettner,K. and Maentselae,P. (1990) Structure of the β-1,3–1,4-glucanase gene of Bacillus macerans: homologies to other β-glucanases. Mol. Gen. 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 Genet., 222, 278–283. µ Christensen,T., Woldike,H., Boel,E., Mortensen,S.B., Hjortshoj,K., Thim,L. and AMX600 spectrometer at 600 MHz and 298 K. Then 20 l of an Hansen,M.T. (1988) High level expression of recombinant genes in Aspergillus XEG or cellulase (Megazyme) solution (20 units, in D2O oryzae. Bio/Technology, 6, 1419–1422.

99 M.Pauly et al.

Claeyssens,M. and Henrissat,B. (1992) Specificity mapping of cellulolytic Sakamato,S., Tamura,G., Ito,K., Ishikawa,T., Iwano,K. and Nishiya,N. (1995) enzymes: Classification into families of structurally related proteins confirmed by Cloning and sequencing of cellulase cDNA from Aspergillus kawachii and its biochemical analysis. Protein Sci., 1, 1293–1297. expression in Saccharomyces cerevisae. Curr. Genet., 27, 435–439. Dalboge,H., Andersen,L.N., Kofod,L.V., Kauppinen,S. and Christgau,S. (1994) Sambrook,J., Fritsch,E.F. and Maniatis,T. (1997) Molecular Cloning: A Laboratory An enzyme with endoglucanase activity. Patent WO 94/14953. Manual. Cold Spring Harbor, NY. Dalboge,H. and Heldt-Hansen,H.P. (1994) A novel method for efficient Sanger,F., Nicklen,S. and Coulson,A.R. (1977) DNA sequencing with chain- expression cloning of fungal enzyme genes. Mol. General Genet., 243, 253–260. terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463–5467. Devereux,J., Haeberli,P. and Smithies,O. (1984) A comprehensive set of sequence Schou,C., Rasmussen,G., Kaltoft,M.-B., Henrissat,B. and Schülein,M. (1993) analysis programs for the VAX. Nucleic Acids Res., 12, 387–395. Stereochemistry, specificity and kinetics of the hydrolysis of reduced Edwards,M., Dea,I.C.M., Bulpin,P.V. and Reid,J.S.G. (1985) Xyloglucan (amyloid) cellodextrins by nine . Eur. J. Biochem., 217, 947–953. mobilisation in the cotyledons of Tropaeolum majus L. seeds following Sherman,F. (1991) Getting started with with yeast. Methods Enzymol., 194, 3–21. germination. Planta, 163, 133–140. Sims,I.M., Munro,S.L.A., Currie,G., Craik,D. and Bacic,A. (1996) Structural Edwards,M., Dea,I.C.M., Bulpin,P.V. and Reid,J.S.G. (1986) Purification and characterisation of xyloglucan secreted by suspension-cultured cells of properties of a novel xyloglucan-specific endo- (1→4)-β-D-glucanase from Nicotiana plumbaginifolia. Carbohydr. Res., 293, 147–172. germinated nasturtium seeds (Tropaeolum majus L). J. Biol. Chem., 261, Sinnott,M.L. (1990) Catalytic mechanisms of enzymatic glycosyl transfer. Chem. 9489–9494. Rev., 90, 1171–1202. Fanutti,C., Gidley,M.J. and Reid,J.S.G. (1993) Action of a pure xyloglucan Strathern,J.N. and Higgins,D.R. (1991) Recovery of plasmids from yeast into endo-transglycosylase (formerly called xyloglucan-specific Escherichia coli: shuttle vectors. Methods Enzymol., 194, 319–329. endo-(1→4)-β-D-glucanase) from the cotyledons of germinated nasturtium Tomme,P., Van Tilbeurgh,H., Pettersson,G., Van Damme,J., Vandekerckhove,J., seeds. Plant J., 3, 691–700. Knowles,J., Teeri,T. and Claeyssens,M. (1988) Studies of the cellulolytic Friebolin,H. (1991) Basic one- and two-dimensional NMR spectroscopy, VCH, system of Trichoderma reesei QM 9414. Analysis of domain function in two Weinheim. cellobiohydrolases by limited proteolysis. Eur. J. Biochem., 170, 575–581. Fry,S.C., Smith,R.C., Renwick,K.F., Martin,D.J., Hodge,S.K. and Matthews,K.J. Tomme,P., Warren,R.A.J. and Gilkes,N.R. (1995) Cellulose hydrolysis by (1992) Xyloglucan endotransglycosylase, a new wall-loosening enzyme bacteria and fungi. Adv. Microbial Physiol., 37, 1–81. activity from plants. Biochem. J., 282, 821–828. Torronnen,A., Kubicek,C.P. and Henrissat,B. (1993) Amino acid sequence β Guillen,R., York,W.S., Pauly,M., An,J., Impallomeni,G., Albersheim,P. and similarities between low molecular weight endo-1,4- -xylanases and family Darvill,A. (1995) Metabolism of xyloglucan generates xylose-deficient H cellulases revealed by clustering analysis. FEBS Lett., 23, 135–139. oligosaccharide subunits of this polysaccharide in etiolated peas. Carbohydr. van den Hazel,H.B., Kielland-Brandt,M.C. and Winther,J.R. (1992) Autoactivation Res., 227, 291–311. of proteinase A initiates activation of yeast vacuolar zymogens. Eur. J. Hayashi,T. (1989) Xyloglucans in the primary cell wall. Annu. Rev. Plant Physiol. Biochem., 207, 277–283. Plant Mol. Biol., 40, 139–168. Verma,D.P.S., Maclachlan,G., Byrne,H. and Ewings,D. (1975) Regulation and in Hayashi,T., Marsden,M.P.F. and Delmer,D.P. (1987) Pea xyloglucan and cellulose. 5. vitro translation of messenger ribonucleic acid for cellulase from auxin-treated Xyloglucan-cellulose interactions in vitro and in vivo. Plant Physiol., 83, pea epicotyls. J. Biol. Chem., 250, 1019–1026. Vincken,J.-P., Beldman,G. and Voragen,A.G.J. (1994) The effect of xyloglucans 384–389. on the degradation of cell-wall embedded cellulose by the combined action of Henrissat,B. and Bairoch,A. (1993) New families in the classification of glycosyl cellobiohydrolase and endoglucanases from Trichoderma viride. Plant hydrolases based on amino acid sequence similarities. Biochem. J., 293, Physiol., 104, 99–107. 781–788. Vincken,J.P., Beldman,G. and Voragen,A.G.J. (1997) Substrate specificity of Kiefer,L.L., York,W.S., Darvill,A.G. and Albersheim,P. (1989) Structure of plant endoglucanases: What determines xyloglucanase activity? Carbohydr. Res., cell walls. 27. Xyloglucan isolated from suspension-cultured sycamore cell 298, 299–310. walls is O-acetylated. Phytochemistry, 28, 2105–2107. Ward,M., Wu,S., Dauberman,J., Weiss,G., Larenas,E., Bower,B., Rey,M., Kitamato,N., Go,M., Shibayama,T., Kimura,T., Kito,Y., Ohmiya,K. and Clarkson,K. and Bott,R. (1993) Cloning, sequence and preliminary structural Tsukagoshi,N. (1996) Molecular cloning, purification and characterization of β analysis of a small, high pI endoglucanase (EG III) from Trichoderma viride. two endo-1,4- -glucanases from Aspergillus oryzae KBN616. Appl. Microbiol. In Suominen,P. and Reinikainen,T. (eds.), Proceedings of the Second TRICEL Biotechnol., 46, 538–544. Symposium on Trichoderma reesei Cellulases and Other Hydrolases, Espoo, Kofod,L.V., Kaupinnen,S., Christgau,S., Andersen,L.N., Heldt-Hansen,H.P., Finland, pp. 153–157. Doerreich,K. and Dalboge,H. (1994) Cloning and characterization of two Wilder,B.M. and Albersheim,P. (1973) The structure of plant cell walls. 4. structurally and functionally divergent rhamnogalacturonases from Aspergillus A structural comparison of the wall hemicellulose of cell supspension cultures aculeatus. J. Biol. Chem., 269, 29182–29189. of sycamore (Acer pseudoplatanus) and of red kidney bean (Phaseolus Kooiman,P. (1961) The constitution of Tamarindus-amyloid. Rec. Trav. Chim., vulgaris). Plant Physiol., 51, 889–893. 80, 849–865. Wittmann,S., Shareck,F., Kluepfel,D. and Morosoli,R. (1994) Purification and Lever,M. (1972) A new reaction for colorimetric determination of carbohydrates. characterization of the Cel B endoglucanase from Streptomyces lividans 66 Anal.Biochem., 47, 273–279. and DNA sequence of the encoding gene. Appl. Environ. Microbiol., 60, Maruyama,K., Goto,C., Numata,M., Suzuki,T., Nakagawa,Y., Hoshino,T. and 1701–1703. Uchiyama,T. (1996) O-acetylated xyloglucan in extracellular polysaccharides Xu,W., Purugganan,M.M., Polisensky,D.H., Antosiewicz,D.M., Fry,S.C. and from cell-suspension cultures of Mentha. Phytochemistry, 41, 1309–1314. Braam,J. (1995) Arabidopsis TCH4, regulated by hormones and the Matsumoto,T., Sakai,F. and Hayashi,T. (1997) A xyloglucan-specific environment, encodes a xyloglucan endotransglycosylase. Plant Cell, 7, endo-1,4-β-glucanase isolated from auxin-treated pea stems. Plant Physiol., 1555–1567. 114, 661–667. York,W.S., Darvill,A.G. and Albersheim,P. (1984) Inhibition of 2,4-dichloro- Murao,S., Sakamato,S. and Arai,M. (1988) Cellulases of Aspergillus aculeatus. phenoxyacetic acid-stimulated elongation of pea stem segments by a Methods Enzymol., 160, 274–299. xyloglucan oligosaccharide. Plant Physiol., 75, 295–297. Ooi,T., Shinmyo,A., Okada,H., Hara,S., Ikenaka,T., Murao,S. and Arai,M. York,W.S., Harvey,L.K., Guillen,R., Albersheim,P. and Darvill,A.G. (1993) (1990a) Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) Structural analysis of tamarind seed xyloglucan oligosaccharides using from Aspergillus aculeatus. Curr. Genet., 18, 217–222. β-galactosidase digestion and spectroscopic methods. Carbohydr. Res., 248, Ooi,T., Shinmyo,A., Okada,H., Murao,S., Kawaguchi,T. and Arai,M. (1990b) 285–301. Complete nucleotide sequence of a gene coding for Aspergillus aculeatus York,W.S., Kolli,V.S.K., Orlando,R., Albersheim,P. and Darvill,A.G. (1996) The cellulase (FI-CMCase). Nucleic Acids Res., 18, 5884. structures of arabinoxyloglucans produced by solaneceous plants. Carbohydr. Perito,B., Hanhart,E., Irdani,T., Iqbal,M., McCarthy,A.J. and Mastromei,G. Res., 285, 99–128. (1994) Characterization and sequence analysis of a Streptomyces rochei A2 York,W.S., Oates,J.E., van Halbeek,H., Darvill,A.G. and Albersheim,P. (1988) endoglucanase-encoding gene. Gene, 148, 119–124. Structure of plant cell walls. 21. Location of the O-acetyl substituents on a Saarilahti,H.T., Henrissat,B. and Palva,E.T. (1990) CelS: a novel endoglucanase nonasaccharide repeating unit of sycamore extracellular xyloglucan. Carbohydr. identified from Erwinia carotovora subsp. carotophora. Gene, 90, 9–14. Res., 173, 113–132.

100