Glycobiology

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Glycobiology, 2000, Vol. 10, No. 2 193-201
© 2000 Oxford University Press

Preparation of oligomeric ß-glycosides from cellulose and hemicellulosic polysaccharides via the glycosyl transferase activity of a Trichoderma reesei cellulase

William S. York¹ and Robert Hawkins

Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602–4712, USA

Received on June 1, 1999; revised on August 13, 1999; accepted on August 19, 1999.

	Abstract

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
Oligoglycosyl (allyl, 2,3-dihydroxypropyl, ethyl, 2-hydroxyethyl, and methyl) ß-glycosides were generated by endo-transglycosylation reactions catalyzed by commercially available Trichoderma reesei cellulase. A polymeric donor substrate (xyloglucan or cellulose) was incubated with the enzyme in an aqueous solution containing 20% of the acceptor alcohol (allyl alcohol, glycerol, ethanol, ethylene glycol, and methanol, respectively). The products of these reactions included oligomeric alkyl ß-glycosides and reducing oligosaccharides. The high yield of alkyl ß-glycosides may be explained by the resistance of the xyloglucan ß-glycosides to cellulase-mediated hydrolysis. The resistance of the oligoxyloglucan ß-glycosides to endoglucanase catalyzed hydrolysis supports the hypothesis that productive binding of the glycan substrate depends on its interaction with enzyme subsites on both sides of the cleavage point, leading to distortion of the ring geometry of the residue whose glycosidic bond is cleaved. Oligoxyloglucan ß-glycosides were purified by a combination of gel-permeation and reversed-phase HPLC and were structurally characterized by MS and NMR spectroscopy. These results demonstrate that novel oligosaccharide ß-glycosides can be efficiently produced by enzyme-catalyzed fragmentation/transglycosylation reactions starting with a polysaccharide donor substrate. This class of reactions may represent a convenient source of ß-glycosides to be used as synthons for the rapid synthesis of complex glycans.

Key words: endoglucanase/transglycosylation/ß-glycoside/xyloglucan/cellulose

	Introduction

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
Enzymes that normally hydrolyze glycosidic bonds are extremely useful catalysts for the in vitro synthesis of oligo­saccharide (Crout and Vic, 1998; Carbohydrate Research, 1998). Two classes of synthetic reactions are catalyzed by these enzymes (Crout and Vic, 1998) under the appropriate conditions. (1) Reverse hydrolysis occurs when the hydrolase is incubated with high concentrations of the products (monosaccharides, oligosaccharides, and/or alcohols) that are generated by the enzyme under "normal" conditions (i.e., those that favor hydrolysis.) Enzyme-mediated catalysis facilitates the approach to thermodynamic equilibrium, so the relative rates of hydrolysis and reverse hydrolysis can be controlled by changing the relative concentration of the reactants. Reverse hydrolysis is favored when the concentration of water is low and the concentrations of other reactants are high. (2) The kinetic approach to glycoside synthesis exploits the capacity of a hydrolase to catalyze the transfer of glycosidic bonds. Hydrolysis involves cleavage of the bond between the glycosidic carbon and the glycosidic oxygen, and formation of a bond between the glycosidic carbon and the oxygen atom of a water molecule. Thus, hydrolysis can be thought of as a type of glycosyl transfer reaction in which water acts as the acceptor substrate. Addition of an alternative acceptor substrate that effectively competes with water results in the formation of new glycosidic bonds. As is the case for reverse hydrolysis, the formation of glycosidic bonds is promoted when the activity of water is reduced to a level just above that at which the enzyme fails as a catalyst. However, the yield of a transglycosylation reaction is particularly dependent on the kinetics of the reaction (i.e., the relative rates of competing reactions), whereas the yield of a reverse hydrolysis reaction depends on the thermo­dynamics of the system (i.e., the relative concentrations of products and reactants at equilibrium).

The transglycosylation of polysaccharides is a biologically important process in vivo. For example, xyloglucan (XG) endotransglycosylases cleave and religate the polysaccharides that cross-link the walls surrounding rapidly expanding cells in flowering plants (Fry et al., 1992). Transglycosylation reactions have also been widely used to prepare glycosides in vitro (Crout and Vic, 1998; McCleary, 1999). Typically an exoglyco­sidase is incubated with a low molecular weight donor substrate, such as an aryl glycoside or a glycosyl halide, and a suitable acceptor substrate. Recently, the capacity of an endo­glycosidase to catalyze the in vitro transfer of a decasaccharide from a naturally occurring glycopeptide to a synthetic N-acetylglucosylaminyl peptide acceptor substrate has been demonstrated (Yamomoto et al., 1998). Endoglucanases (EGs) have also been used to catalyze transglycosylation reactions in which the donor substrate is a diglycosyl halide (e.g., lactosyl fluoride) and the acceptor substrate is another oligosaccharide (Moreau and Driguez, 1996; Armand et al., 1997). However, endoglycosidases have not previously been used to prepare glycosides via transglycosylation reactions in which a polysaccharide is used as the donor substrate.

Glycosyl hydrolases are commonly used to catalyze the formation of a glycosidic bond between two sugars, that is, via reactions in which the acceptor substrate is itself a carbo­hydrate. However, the products of reactions in which the acceptor substrate is a small alcohol are also extremely useful. For example, allyl glycosides are versatile reagents for the chemical synthesis of complex carbohydrate structures. Isomerization of the allyl aglycon generates a highly reactive vinyl glycoside (Marra et al., 1992; Boons and Isles, 1996), which can be used as a reagent in chemically catalyzed glycosylation reactions. Hydrolase-catalyzed transglycosylation and reverse-hydrolysis reactions have been used (Vic and Crout, 1995; Gibson et al., 1997) to produce allyl and butenyl glycosides for this purposes, but the transglycosylation products produced thus far have consisted of only one or two sugars and the reactive aglycon.

This paper describes the first use of a polysaccharide as a donor substrate for a preparative glycosyltransferase reaction. The use of polysaccharide donor substrates has great potential for the rapid and economic production of useful glycosides. Polysaccharides produced by plants and microbes represent a wide range of structural motifs and are often easy to prepare in large quantities. For example, the bacterial polysaccharide xanthan and the plant polysaccharide cellulose are routinely produced by the ton.

Another important class of polysaccharides are the hemicellulosic XGs, which are major constituents of the cell walls of higher plants (York et al., 1990, 1993, 1996). The XG backbone is composed of ß-(14)-linked D-Glcp residues, up to 75% of which are branched, bearing a sidechain at O-6 (Figure 1). The structures of the XG sidechains vary in different plant tissues and species (York et al., 1990, 1996), the most common being: (1) -D-Xylp-; (2) ß-D-Galp-(12)--D-Xylp-; (3) -L-Fucp-(12)-ß-D-Galp-(12)--D-Xylp-; and (4) -L-Araf-(12)--D-Xylp-. EGs from several sources selectively hydrolyze the unbranched ß-(14)-linked D-Glcp residues in the backbone of the XG, generating a mixture of biologically active xyloglucan oligosaccharides (XGOs, Figure 1) (York et al., 1984). This report describes EG-catalyzed transglyco­sylation reactions in which the donor substrate is either cellulose or XG. These reactions can be used to prepare relatively large and complex oligoglycoside products that may be useful in themselves or may be used as synthons for the production of more complex structures by chemical methods.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Structures of the four major xyloglucan oligosaccharides (XGOs) produced by EG-treatment of tamarind xyloglucan. The sequences of XGOs and oligoglycosides are indicated (Fry et al., 1993) by a series of uppercase letters, each of which represent the branching pattern of an individual ß-D-Glcp residue in the backbone. Each code letter represents the entire substructure, including both the side chain(s) and the backbone ß-D-Glcp residue itself, as illustrated here. The structure of a XGO is thus written by starting at the nonreducing end and sequentially listing the code letters that represent the patterns of side chain substitution for the ß-D-Glcp residues in the backbone. The aglycon of each ß-glycoside described in this article is indicated as the rightmost element of this list. Thus, the oligoglycoside -D-Xylp-(16)-ß-D-Glcp-(14)-[-D-Xylp-(16)-]-ß-D-Glcp-(14)-[-D-Xylp-(16)-]-ß-D-Glcp-(14)-ß-D-Glcp-(11')-OCH3 is indicated by the sequence XXXG-Me. In addition, each ß-D-Glcp residue and the glycosyl residues of its pendant side chain are distinguished by a lower case (superscript) letter that indicates the location of the ß-D-Glcp residue vis-à-vis the aglycon, as indicated at the top of this figure and described in the footnotes of Table II.

 

	Results

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
Generation of ß-alkyl oligoglycosides from XG
Ethanol (EtOH) precipitation is a convenient method to prepare XG from the water-soluble material extracted from tamarind seed meal (Kooiman, 1961). In an attempt to streamline the preparation of oligosaccharides from tamarind XG, the precipitated polysaccharide, still containing residual EtOH, was immediately treated with a commercially available EG (see Materials and methods). Matrix assisted, laser-desorption ionization with time-of-flight (MALDI-TOF) mass spectro­metry of the products indicated that the reaction generated the expected hydrolysis products (Glc₄Xyl₃, [M+Na]⁺ at m/z = 1086; [M+K]⁺ at m/z = 1102; Glc₄Xyl₃Gal, [M+Na]⁺ at m/z = 1248, [M+K]⁺ at m/z = 1264; and Glc₄Xyl₃Gal₂, [M+Na]⁺ at m/z = 1410, [M+K]⁺ at m/z = 1426) along with transglycosylation products (the ethyl glycosides Glc₄Xyl₃-OCH₂CH₃, [M+Na]⁺ at m/z = 1114; [M+K]⁺ at m/z = 1130; Glc₄Xyl₃Gal-OCH₂CH₃, [M+Na]⁺ at m/z = 1276, [M+K]⁺ at m/z = 1292; Glc₄Xyl₃Gal₂-OCH₂CH₃; [M+Na]⁺ at m/z = 1438, [M+K]⁺ at m/z = 1454). The 1-D ¹H-NMR spectrum of the reaction products also included the O-ethyl CH₃ resonance (dd, 1.232, ³J_1,2 = 6.9 Hz), and a resonance (d, 4.497, ³J_1,2 = 8 Hz), assigned as H1 of the 1-O-ethyl ß-Glcp residues. The H1 resonances (d, 5.223, ³J_1,2 = 4 Hz, and d, 4.664, ³J_1,2 = 8 Hz) of the - and ß- reducing glucose residues (Pauly et al., 1999) were also observed.

Several other alcohols were screened for their suitability as acceptor substrates for the EG-catalyzed glycosyl transfer reaction (Table I). The MALDI-TOF mass spectra of the products obtained when tamarind XG was treated in the presence of various alcohols are shown in Figure 2. Some of the alcohols, most notably those containing a sulfhydryl group, inhibit the catalytic activity of the EG, and no depolymerization products are observed in the mass spectra of the treated material. Isopropanol, a secondary alcohol, appeared to be a much poorer acceptor substrate than the homologous primary alcohol n-propanol. However, trans-1,2-cyclohexanediol, which contains only secondary alcohols, is an efficient acceptor substrate; (1R,2R)-trans-1,2-cyclohexanediol, in which the oxygen-bearing carbons have the same absolute stereochemistry as C3 and C4 of a glucopyranosyl residue, is a better acceptor than the 1S,2S isomer. Saligenin (o-hydroxybenzyl alcohol) is not soluble in H₂O at a concentration of 20%, and so this acceptor was included in the reaction mixture as a suspension. Nevertheless, ~25% of the products generated under these conditions were saligenin glycosides. It was not determined which of the two hydroxyl groups in saligenin was more reactive.

View this table: [in this window] [in a new window]   Table I. Endoglucanase-catalyzed glycosyl transfer to various acceptor-substrate alcohols

 

View larger version (28K): [in this window] [in a new window]   Fig. 2. MALDI-TOF mass spectra of the mixtures of products generated by treating tamarind XG with EG in the presence of various alcohols. The m/z ratios for [M + Na]+ ions are indicated. [M + K]+ ions were also detected, but not labeled. Digestion in the absence of acceptor alcohol (i.e., in 100% H2O) generated the expected oligosaccharides XXXG (m/z 1086), XXLG/XLXG (m/z 1248), and XLLG (m/z 1410). These reducing oligosaccharides were also detected when the reaction was carried out in the presence of various acceptor alcohols, along with the corresponding ß-glycosides (indicated by an asterisk) generated by transglycosylation. (1R,2R)-trans-1,2-cyclohexanediol is abbreviated RR-CHD, and saligenin is o-hydroxybenzyl alcohol.

 
Purification and characterization of ß-alkyl oligoglycosides from XG
Additional EG-catalyzed transglycosylation reactions were carried out using tamarind XG as a donor substrate in 20% aqueous alcohol (ethanol, glycerol, and methanol). SEC on Bio-Gel P-10 was used to remove the products (MW > 2,000) resulting from incomplete cleavage of the XG (data not shown). The Bio-Gel P-10 fractions containing the low-molecular weight products, which accounted for 40–90% of digested material, were pooled, concentrated, and separated by SEC on Bio-Gel P-2. The chromatogram of the products obtained when the transglycosylation reaction was carried out in 20% methanol is illustrated in Figure 3. MALDI-TOF analysis indicated that the major components of Peaks 1 and 2 were methyl oligoglycosides Glc₄Xyl₃Gal₂OCH₃ ([M+Na]⁺ at m/z = 1424) and Glc₄Xyl₃GalOCH₃ ([M+Na]⁺ at m/z = 1262), respectively. Peak 3 contained a mixture of the reducing nonasaccharide Glc₄Xyl₃Gal₂ ([M+Na]⁺ at m/z = 1410) and the methyl heptaglycoside Glc₄Xyl₃OCH₃ ([M+Na]⁺ at m/z = 1100). The major components of Peaks 4 and 5 were the reducing oligosaccharides Glc₄Xyl₃Gal ([M+Na]⁺ at m/z = 1448) and Glc₄Xyl₃ ([M+Na]⁺ at m/z = 1086), respectively. Thus, even the presence of a small methyl aglycon has a significant effect on the chromatographic behavior of a XG oligoglycoside during SEC. This made it possible to obtain fractions highly enriched in the ß-alkyl nonaglycosides XLLG-Et, XLLG-gro, and XXLG-Me (Figure 4A–C) by Bio-Gel P-2 chromato­graphy. Due to the non-polar character of the methyl aglycon, the ß-methyl glycosides XLXG-Me, XXLG-Me, and XXXG-Me could be separated from reducing oligosaccharides by reversed-phase HPLC of individual P-2 fractions on octadecyl silica (ODS). Under the conditions used, the reducing XG oligomers were eluted between 23 and 33 min. The four most abundant methyl XG glycosides were eluted at 47.3 min (XLXG-Me), 47.9 min (XXXG-Me), 50.0 min (XLLG-Me), and 53.0 min (XXLG-Me).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Bio-Gel P-2 chromatography of low molecular weight products generated by treating tamarind XG with EG in the presence of 20% MeOH. Carbohydrate was detected by the anthrone assay (absorbance at 620 nm). The major components contributing to each peak of anthrone-positive material are: (1) XLLG-Me; (2) XXLG-Me and XLXG-Me; (3) XLLG and XXXG-Me; (4) XXLG and XLXG; (5) XXXG (see Materials and methods for a description of this nomenclature.) Similar chromatograms were obtained upon P-2 chromatography of the products generated by transglycosylation to other alcohols, including ethanol and glycerol.

 

View larger version (26K): [in this window] [in a new window]   Fig. 4. Anomeric regions of the 1H-NMR spectra of ß-alkyl oligoglycosides generated by EG-catalyzed transglycosylation reactions using tamarind XG as the donor substrate. (See Materials and methods for a description of the nomenclature used.) Glycosides were purified by liquid chromatography on Bio-Gel P-2 (see Figure 1). Methyl glycosides (C-F) were further purified by reverse-phase chromatography on octadecyl silica.

 
The HPLC-purified alkyl XG oligoglycosides were identified by MALDI-TOF analysis and by comparing their 1-D ¹H-NMR spectra (Figure 4) to spectra of tamarind XG oligosaccharides and oligoglycosyl alditols whose structures have been rigorously established (Kooiman, 1961; York et al., 1990, 1993). The structures of the P-2 purified XLLG-gro and the HPLC purified XXXG-Me and XXLG-Me were confirmed by 2-D NMR (COSY, NOESY, TOCSY, HSQC, and HMBC), which made it possible to assign every resonance in their ¹H- and ¹³C-NMR spectra to a specific spin system (Tables II, III). The assigned sugar geometries and anomeric configurations were consistent with the observed ¹H-¹H scalar coupling constants.

View this table: [in this window] [in a new window]   Table II. 1H-NMR assignments for the oligoglycosides

 

View this table: [in this window] [in a new window]   Table III. 13C-NMR assignments for the oligoglycosides

 
Complete assignment of the NMR spectra of XXLG-Me was complicated by the fact that the anomeric protons of two of the ß-Glcp residues and the ß-Galp residue are isochronous at 4.555 (Figure 4D). Nevertheless, sequence-specific assignments were made starting with the resolved H1 resonances ( 4.406 and 4.550) of two of the ß-Glcp residues, designated Glc^g and Glc^c, respectively. (See Figure 1 and footnotes to Table II for a description of this nomenclature.) H1 of Glc^g ( 4.406) exhibits an NOE contact with the CH₃ ( 3.571) protons of the O-methyl aglycon and a long range heteronuclear coupling to the O-methyl carbon ( 58.0). The chemical shift of C6 of Glc^g ( 60.9) indicates that this residue does not bear a substituent at C6. H1 of an -Xylp residue ( 4.941) exhibits an NOE contact with H6 (Pro-S, 3.780) of Glc^c and a heteronuclear coupling to C6 ( 66.7) of Glc^c, indicating that Glc^c bears a terminal -Xylp residue (i.e., Xyl^c) at C6. No NOE contacts or heteronuclear couplings were observed that might suggest that Glc^c bears a glycosyl substituent at C4 ( 70.3). H1 of Glc^c has an NOE contact with H4 ( 3.734) and a hetero­nuclear coupling to C4 ( 80.2) of one of the ß-Glcp residues (Glc^b) whose anomeric proton resonance ( 4.555) is not resolved. However, H1 of an -Xylp residue ( 4.959) has an NOE contact with H6 (Pro-S, 3.898) of Glc^b and a hetero­nuclear coupling with C6 ( 66.9) of Glc^b, indicating that Glc^b also bears a terminal -Xylp residue (i.e., Xyl^b) at C6. A broad, intense crosspeak in the NOESY spectrum correlates the unresolved ß-anomeric protons at 4.555 and several other protons at 3.66–3.76. Due to severe signal overlap, it is not possible to extract a great deal of structural information from this crosspeak. However, heteronuclear couplings between H4 of Glc^g ( 3.675) and C1 of Glc^a ( 103.2) and between H4 of Glc^a ( 3.668) and C1 of Glc^b ( 103.4) were observed. The combined NMR data is consistent with the glycosyl sequence Glc^cGlc^bGlc^aGlc^gMe for the backbone of the octaglycoside. The data also indicate that monoglycosyl (-D-Xylp-) sidechains are attached to C6 of Glc^c and Glc^b, and a diglycosyl (ß-D-Galp-(12)--D-Xylp-) sidechain is attached to C6 of Glc^a. These results are completely consistent with the initial structural assignments for the methyl oligo­glycosides that were based on structurally diagnostic chemical shift effects previously observed in the NMR spectra of well characterized XG oligosaccharides and oligoglycosyl alditols (York et al., 1990, 1993).

The ß-Glcp residue of XLLG-gro bearing the glyceryl aglycon was identified by a long-range heteronuclear coupling from its anomeric carbon (C1 103.0) to two geminal protons (H1, 3.759 and H1' 3.913) of the glycerol moiety. This assignment was confirmed by the observation of NOESY crosspeaks between H1 ( 4.504) of this ß-Glcp residue and the same two geminal protons. These results indicate that the transglycosylation product is a ß-glycoside linked to one of the primary hydroxyl groups of the glycerol moiety, as expected, based on the greater reactivity of n-propanol relative to iso-propanol (see above). The absolute stereochemistry of the glycerol (sn-1-linked or sn-3-linked) in this major product was not determined.

Preparation of ß-methyl cellobiosides from cellulose
EG-treatment of cellulose in the presence of methanol as an acceptor substrate resulted in the generation of cellobiose, glucose, ß-methyl cellobioside, and ß-methyl glucoside. The EG-catalyzed depolymerization of insoluble cellulose is a heterogeneous reaction, and only a small proportion (usually less than 5%) of the cellulose was solubilized under the conditions used. However, up to 50% of the soluble products were recovered as ß-glycosides. Reconstituted, hydrated cellulose (i.e., dialysis tubing), appeared to be a better substrate than crystalline cellulose. EG-treatment of Sigmacell 100 generated several unidentified products in addition to cellobiose, glucose, ß-cellobiosides, and ß-glucosides, which were the dominant products generated when Avicel or dialysis tubing was used as a substrate. The presence of 20% methanol did not significantly inhibit the capacity of the EG to solubilize cellulose, and yields were not linearly dependent on the amount of EG or cellulose used in the reaction (Table IV). In general, the absolute amount of EG-solubilized carbohydrate increased ~4-fold when the amount of cellulose substrate was increased 10-fold (all other conditions being kept equal). The absolute amount of EG-solubilized carbohydrate increased ~2-fold when the amount of EG was increased 10-fold. As cellulose is much less expensive than EG, the most cost-effective implementations of this reaction will likely involve conditions that solubilize only a small portion of the cellulose. Pretreatment of the cellulose with a swelling agent to make it more accessible to the enzyme may also be advantageous.

View this table: [in this window] [in a new window]   Table IV. Amount of cellulose solubilizeda by EG in 20% MeOH and 100% H2O

 
Resistance of alkyl ß-glycosides to hydrolysis by the EG
Several experiments were carried out to determine whether the alkyl ß-glycoside products produced by EG-catalyzed trans­glycosylation reactions could themselves act as glycosyl donor substrates. For example, aqueous solutions containing high concentrations of ß-glyceryl XG oligoglycosides were incubated with the EG. The products were analyzed to determine whether larger oligoglycosides were generated under these conditions, which would indicate that the oligoglycosides could act as both donor and acceptor substrates for the transglycosylation reaction. However, the ß-glyceryl XG oligoglycosides were not modified in any detectable way by the EG, indicating that the EG was not capable of cleaving the glycosidic bond linking the glyceryl moiety to the oligosaccharide (data not shown). Further experiments were performed in which aqueous solutions of highly purified ß-glyceryl and ß-ethyl XG oligoglycosides were incubated with a large excess of the EG and the products were analyzed by MALDI-TOF MS. Although the amount of EG that was added could theoretically hydrolyze the ß-glycosides several hundred times during the incubation period (based the enzyme activity toward polymeric XG), no hydrolysis products were detected. This indicates that the EG and the alkyl ß-glycosides do not interact to form a complex that leads to the hydrolysis of the glycosidic bond to the alkyl group.

	Discussion

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
A broad range of small alcohols are efficiently used as acceptor substrates for EG-catalyzed transglycosylation (Table I). It should be possible to use several of the ß-glycoside products listed in Table I as reagents to produce a range of structurally diverse products. For example, reaction of a nucleophile with the keto group of the ß-glycoside of 4-hydroxy-3-methyl-butanone could be exploited to link the glycoside to a polypeptide or other noncarbohydrate moiety. Allyl glycosides, which were produced with high efficiency (Table I) can be oxidized (after protection of the sugar-hydroxyl groups) to produce glycosides with a reactive aldehyde in the aglycon (Díaz et al., 1998), or isomerized to form vinyl glycosides, which are versatile reagents used in chemically catalyzed glycosylation reactions (Marra et al., 1992; Boons and Isles, 1996).

It is, at first glance, somewhat surprising that the ß-glycosides generated by EG-catalyzed trans­glycosylation reactions are resistant to hydrolysis by the EG (Crout and Vic, 1998), as it is often postulated that enzymes catalyze the forward and reverse reactions with equal efficiency and that the distribution of products depends on their relative thermodynamic stabilities.

The resistance of the ß-glycosides to EG-catalyzed hydrolysis is consistent with the catalytic mechanism proposed (Sulzenbacher et al., 1996) for Family 7 EGs (Figure 5), such as the Megazyme endoglucanase (EG-1 from Trichoderma reesei) used in this study. According to this model, binding of a substrate distorts the geometry of the glucosyl residue in the –1 subsite, placing the potential leaving group in the axial orientation, consistent with the proposed geometry of the transition state. The distorted axial geometry of the glycosidic oxygen is energetically unstable in aqueous solution, where the glycosidic oxygens of ß-glucosyl residues are invariably found in the equatorial orientation. Therefore, the increase in free energy due to the distortion must be compensated by a decrease in free energy due to the association of the glucan substrate with the EG. Presumably, the distortion of the glucosyl residue in the -1 subsite of the EG depends, in part, on the strong interaction of the adjacent glucosyl residue with the +1 binding subsite of the EG, leading to a strained complex whose total free energy is lower than that of the isolated enzyme and substrate (Sulzenbacher et al., 1996). Stabilization of the transition state facilitates the formation of a enzyme substrate intermediate in which the glucosyl residue is covalently attached to the sidechain of a glutamic acid. During hydrolysis reactions, this intermediate is attacked by a water molecule, releasing the product as an oligosaccharide whose reducing glucose residue is in the ß-configuration (i.e., with retention of anomeric configuration). During transglycosylation reactions, the intermediate is attacked by an alcohol, releasing the product as a ß-glycoside. The resistance of the alkyl ß-glycosides to hydrolysis by the EG is consistent with this scenario, in that the aglycon moieties of these glycosides are chemically distinct from the glucosyl residues that have a putatively strong interaction with the +1 binding subsite of the EG. Apparently, the weak interaction of the alkyl aglycon with the +1 binding subsite does not provide sufficient energy to induce geometric distortion of the glucosyl residue in the -1 subsite. Therefore, no productive complex is formed, even if the alkyl glycoside happens to bind to the active site of the EG and the alkyl glycoside is not a substrate for the EG.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Proposed mechanism (Sulzenbacher et al., 1996) for the EG-catalyzed hydrolysis and transglycosylation reactions. Binding of the substrate to the active site of the EG (A) results in a distortion of the ß-glucosyl residue in the -1 site, facilitating the development (B) of the transition state, which leads to the formation (C) of a glycosidic bond between the non-reducing-end fragment of the substrate and the enzyme. This covalent complex is attacked (D) by a nucleophile (i.e., water (R' = -H) or an alcohol (R' = -CH3, -CH2CH3, -CH2CH2OH, etc.)), leading to a second transition state that resembles the first. The resulting lysis (E) of the glycosidic bond between the enzyme and the glycan fragment generates a reducing oligosaccharide product when the nucleophile is water or a ß-glycoside when the nucleophile is an alcohol. The product is then released (F) from the enzyme. All steps are likely to be reversible, except step (E) whose reversal is energetically unfavorable because the interaction of the aglycon (i.e., R') with the +1 subsite does not provide sufficient energy to distort the glycosyl residue in the -1 subsite, and facilitate reformation of the transition state.

 
The production of ß-alkyl-oligoglycosides by EG-catalyzed transglycosylation reactions is facilitated by the fact that the products tested thus far are resistant to attack by the EG. This resistance makes it unnecessary to balance forward and backward reaction rates by carefully adjusting the substrate concentrations and incubation times, as the ratio of ß-glycosides to reducing products depends primarily on the competition between transglycosylation and hydrolysis. Once transglycosylation has occurred, extension of the incubation period, adjustment of the incubation temperature, dilution of the reaction mixture with water, or removal of the acceptor-substrate alcohol by evaporation does not lead to hydrolysis of the ß-glycoside products. However, it is possible that the ß-glycoside products could act as donor substrates for other glyco­sidases or transglycosylases (acting by a different catalytic mechanism), leading to other products that cannot be made directly by transglycosylation reactions catalyzed by a Family 7 EG.

	Conclusion

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
A Family 7 EG (EG-1), produced by the cellulolytic fungus Trichoderma reesei, catalyzes the transglycosylation from a cellulosic or hemicellulosic donor substrate to small acceptor substrates to generate ß-glycoside products. Resistance of these products to EG-catalyzed hydrolysis makes it relatively easy to find conditions under which they can be recovered in high yield. A wide range of small alcohols, many of which have useful secondary reactivities, are efficient transglyco­sylation acceptors. These results suggest that enzyme-catalyzed transglycosylation reactions using polysaccharides as donor substrates have great potential as a means to prepare structurally diverse products.

	Materials and methods

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
Enzymes
EG was purchased from Megazyme International Ireland (cat. no. E-CELTR, Cellulase (endo-1,4-ß-glucanase), EC 3.2.1.4). This enzyme is also known as EG-1 (McCleary, 1999), which is a Family 7 EG (Davies and Henrissat, 1995).

SEC
Samples were desalted by concentrating them to 2–5 ml and applying them to a column (2.5 x 45 cm) of Sephadex G-10 (Pharmacia) eluted with deionized water (1 ml/min) The anthrone assay (Dische, 1962) was used to determine the carbohydrate content of the collected fractions (2 ml), and the conductivity of the fractions was measured (model 1052A conductometer, Amber Science) to determine their salt content. Oligoglycosides were separated by SEC on Bio-Gel P chromatography supports (Bio-Rad Laboratories). Samples (2 ml) were injected onto a Bio-Gel P-10 column (1.6 x 75 cm) or two Bio-Gel P-2 columns (1.6 x 80 cm) connected in series, and eluted with deionized water (0.1–0.2 ml/min). The carbohydrate content of the collected fractions (2 ml) was determined by the anthrone assay (Dische, 1962).

Reversed-phase chromatography
Oligoglycoside samples were dissolved in water, and up to 250 µl was injected onto a "semi-preparative" octadecyl silica column (10 x 250 mm, Lichrosorb RP-18, Merck). The oligoglycosides were eluted (3 ml/min) with a linear gradient of aqueous methanol (6–12% v/v over 60 min) delivered with an HPLC gradient module (Bio-Rad Laboratories) controlled by a Bio-Rad model 700 chromatography workstation. Approximately 5% of the eluent was diverted via a T-splitter to an evaporative light scattering detector (SEDEX 55 ELSD, S.E.D.E.R.E., Alfortville, France), and the remaining 95% was collected manually (Pauly and York, 1998).

ß-Alkyl oligoglycosides from XG
XG, from defatted tamarind seed powder (York et al., 1990), was dissolved (1 mg/ml) in NaOAc buffer (50 mM, pH 5) containing various alcohols (20% v/v) and treated with EG (0.4 units/mg of XG). The solution was incubated (23°C, 24 h) and then boiled (15 min) to deactivate the enzyme. Volatile alcohols, when present, were removed by evaporation under reduced pressure. The products were concentrated to a small volume (<3 ml) and desalted by SEC on Sephadex G-10. XGOs with molecular weights less than 2000 (typically 40–90% of the soluble products) were isolated from the desalted product by SEC on Bio-Gel P-10 (data not shown). Oligoglycosides in this low molecular weight fraction were separated by SEC on Bio-Gel P-2 (Figure 3). Individual P-2 fractions (Figure 1) derived from the material generated by EG-treatment of XG in 20% methanol were collected, concentrated, and subjected to reversed-phase HPLC on octadecyl silica, to yield pure ß-methyl oligoglycosides (Figure 4C–F).

ß-Alkyl oligoglycosides from cellulose
Reconstituted cellulose (Spectra-Por 6 dialysis tubing (Spectrum), cut into small pieces) was suspended (3.5 g in 200 ml of 50 mM NaOAc buffer, pH 5) containing methanol (20% v/v). EG (10 units) was added and the suspension was incubated (23°C, 24 h). The addition of EG and incubation were repeated two times, after which the reaction mixture was boiled (30 min) to deactivate the enzyme. Methanol was removed by evaporation under reduced pressure. Insoluble material (i.e., undigested cellulose) was then removed by centrifugation (10,000 x g, 20 min, Beckman JA-10 rotor). The soluble products were concentrated to a small volume (5 ml) and desalted by SEC on Sephadex G-10. ß-Methyl cellobioside and ß-methyl-glucoside were isolated from the desalted material by SEC on Bio-Gel P-2.

MALDI-TOF mass spectrometry
MALDI-TOF mass spectra were recorded using a Hewlett Packard G2025A LD-TOF mass spectrometer operating at an accelerating voltage of 4.75 kV and a source pressure of approximately 3 x 10^–7 torr. Desalted, aqueous samples (1 µl) were mixed with a solution (1 µl) of the ionization matrix (1:1 (v/v) 2,5-dihydroxy benzoic acid (0.2 M in 50% aq. CH₃CN) and 1-hydroxy isoquinoline (0.06 M in 50% aq. CH₃CN)). Analyte and matrix were cocrystallized on the probe by evaporation of the solvent under vacuum. Desorption/ionization was accomplished with a 3 ns pulse ( = 337 nm) from a nitrogen laser.

NMR spectroscopy
ß-Glycosides (1–5 mg) were dissolved in D₂O (1 ml, 99.6 atom % ²H, Cambridge Isotope Laboratories, CIL) and lyophilized to replace exchangeable protons with deuterons. The residues were redissolved in D₂O (99.96% atom % ²H, CIL) and NMR spectra were recorded at 298 K with either a Varian Mercury (300 MHz) or Varian Inova (600 MHz or 800 MHz) spectrometer. Double-quantum filtered COSY (Rance et al., 1983), TOSCY (Bax and Davis, 1985), NOESY (Macura and Ernst, 1980), HSQC (Bodenhausen and Ruben, 1980; Norwood et al., 1990), and HMBC (Bax and Summers, 1986) experiments were recorded at 600 MHz (and 800 MHz for XLLG-gro). Pulsed-field gradients were used for coherence selection during the HSQC and HMBC experiments. Chemical shifts are reported relative to internal acetone (¹H 2.225, ¹³C 33.0).

	Acknowledgments

Top Abstract Introduction Results Discussion Conclusion Materials and methods Acknowledgments References

 
This research was supported in part by the U.S. Department of Energy (DOE)-funded Center for Plant and Microbial Complex Carbohydrates (DE-FG05-93ER20097). The authors wish to thank John Glushka for assistance with the NMR spectroscopy.

	Footnotes

 
¹ To whom correspondence should be addressed

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