Plant Physiol. (1998) 117: 153-163

Rhamnogalacturonan -d-Galactopyranosyluronohydrolase¹
An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin¹

Margien Mutter, Gerrit Beldman, Stuart M. Pitson, Henk A. Schols, and Alphons G.J. Voragen^*

Wageningen Agricultural University, Department of Food Chemistry, Bomenweg 2, 6703 HD Wageningen, The Netherlands (M.M., G.B., H.A.S., A.G.J.V.); and School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney 2052 Australia (S.M.P.)

	ABSTRACT

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A new enzyme, rhamnogalacturonan (RG) -d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing -d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 µm and a maximum reaction rate of 160 units mg¹ was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).

	INTRODUCTION

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GalA is the major constituent sugar of pectins in plant cell walls. Most of the GalA residues are present in the HG regions of pectin. It is hypothesized that in cell wall pectin, HG regions occur interspersed with RG regions, which are rich in neutral sugar side chains (De Vries et al., 1981; Thibault et al., 1993; Schols et al., 1995). Ongoing research on HG-degrading enzymes such as polygalacturonases, pectin lyases, and pectate lyases, has been accompanied in the last decade by an increase in reports on enzymic degradation of the hairy RG regions of pectin. The application of pectin-degrading enzymes in general lies in the fruit and vegetable processing industry, where processing and quality can be improved using these enzymes (Pilnik and Voragen, 1993). Furthermore, interest lies in the field of the enzymic degradation in vivo of HG and RG as a potential source of plant signaling molecules (Van Cutsem and Messiaen, 1994). Purified enzymes have also gained significance as analytical tools in structural studies because of their high specificity (Voragen et al., 1993).

A series of enzymes, all highly specific for hairy RG regions of pectin, have been purified and characterized. These include RG-hydrolase (Schols et al., 1990), RG-acetylesterase (Searle-Van Leeuwen et al., 1992), RG-rhamnohydrolase (Mutter et al., 1994), RG-lyase (Azadi et al., 1995; Mutter et al., 1996), and xylogalacturonan exogalacturonase (Beldman et al., 1996). The current paper describes the purification and characterization of the latest enzyme in this series, named RG-galacturonohydrolase. The mode of action, substrate specificity, and several possible applications of RG-galacturonohydrolase are discussed.

	MATERIALS AND METHODS

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Substrates

View this table: [in this window] [in a new window]   Table I. Explanation of codes of the RG oligosaccharides used in the characterization of RG-galacturonohydrolase GA, -GalA (1,2)-linked to Rha, or GalA at the reducing end; R, -Rha (1,4)-linked to GalA, or Rha at the reducing end; uGA, -us-GalA (1,2)-linked to Rha; G, -Gal (1,4)-linked to Rha.

Linear RG fragments (11-16 in Table I) were kindly provided by Dr. C.M.G.C. Renard (Institut National de la Recherche Agronomique, Nantes, France), and were prepared by controlled acid hydrolysis of sugar-beet cell walls and isolated by ion-exchange chromatography and SEC (Renard et al., 1995, 1998).

Substrates used to determine if saccharidases other than RG-galacturonohydrolase were present in the final purified enzyme fraction included PGA (Fluka Chemie AG, Buchs, Switzerland), methoxylated pectin (DM92.3) prepared in our laboratory according to the method of Van Deventer-Schriemer and Pilnik (1976), larchwood arabino--(1,3)/(1,6)-galactan ("stractan," Meyhall Chemical AG, Kreuzlingen, Switzerland), potato arabino--(1,4)-galactan (isolated from potato fiber according to the work of Labavitch et al. [1976]), a linear arabinan from sugar beet kindly provided by British Sugar (Peterborough, UK), xylan from oat spelts (Koch and Light Ltd., Haverhill, UK), carboxymethylcellulose (Akucell AF type 2805, Akzo, Arnhem, The Netherlands), Avicel cellulose (type SF, FMC, Serva, Heidelberg), and soluble starch (Merck AG, Darmstadt, Germany). The following pnp-glycosides, used for screening of glycosidase side activities of RG-galacturonohydrolase, were obtained from Koch and Light Ltd. and from Sigma: pnp--l-Ara_f, pnp--d-Gal_p, pnp--d-Gal_p, pnp--d-Xyl_p, pnp--d-Xyl_p, pnp--d-Man_p, pnp--d-Man_p, pnp--l-Fuc_p, pnp--d-Fuc_p, pnp--d-Glc_p, pnp--d-Glc_p, pnp--l-Rha_p, pnp--d-Glc_pA, and pnp--d-Gal_pA.

Further substrates used for determination of the substrate specificity of RG-galacturonohydrolase included pectin (DM 35; Obipectin, Bischofszell, Switzerland), an -(1,4)-linked GalA dimer, tetramer, heptamer, a -(4,5)-unsaturated GalA (us-GalA) -(1,4)-linked tetramer (Voragen, 1972; Tjan et al., 1974), and a pectate lyase (from Pseudomonas fluorescens GK5, Rombouts et al., 1978) digest of PGA plus 1 mm CaCl₂.

Enzymic Modification of RG Oligomers

Enzyme Purification

Enzyme Assays

Determination of Side Activities of RG-Galacturonohydrolase

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Influence of pH and Temperature on RG-Galacturonohydrolase

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Other Substrate Degradation Studies

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Determination of Molecular Mass and pI

Determination of the molecular mass of RG-galacturonohydrolase activity using SEC was carried out using a Superose 12 HR 10/30 column (Pharmacia). The column was calibrated with endopolygalacturonase (43 kD), RG-rhamnohydrolase (84 kD), RG-hydrolase (53 kD), and several partially purified proteins with molecular masses of 78, 76, 52, 45, and 32 kD, as characterized by SDS-PAGE. A buffer of 150 mm NaOAc (pH 6.0) was used for elution. Retention of RG-galacturonohydrolase and of RG-rhamnohydrolase on this column was monitored by collecting fractions and determining their activity toward the mixture of oligosaccharides 5 and 6 and the mixture of 3 and 4, respectively, as detected using HPAEC (gradient B).

Preparative IEF was performed using a Rotofor preparative IEF cell (Bio-Rad). Bio-Lyte pH 4.0 to 6.0 was used as ampholyte, and 0.1 m NaOH and 0.1 m H₃PO₄ were used as electrolytes in the cathode and anode chambers, respectively. Partially purified protein fractions (70 mg) containing RG-galacturonohydrolase and RG-rhamnohydrolase were pooled from the SP Sepharose Fast Flow separation column and dialyzed against distilled water. Ampholyte was added (to 1.7% w/v) and the sample was applied to the system. Focusing required 3.5 h at 4°C at 12 W constant power supply, after which the fractions from the 20 compartments were collected immediately. After pH measurement, fractions were screened for RG-galacturonohydrolase and RG-rhamnohydrolase activity on enzymically modified RG oligomers using HPAEC (gradient B).

Stereochemical Course of Hydrolysis

Analytical Methods

HPAEC was performed using a Dionex (Sunnyvale, CA) Bio-LC system equipped with a Dionex CarboPac PA-100 (4 × 250 mm) column and a Dionex pulsed electrochemical detection detector in the PAD mode. Gradients of NaOAc in 100 mm NaOH (1 mL min¹) were used as follows: gradient A, 0 to 7 min, 100 to 200 mm; 7 to 10 min, 200 to 1000 mm; 10 to 15 min, 1000 mm; 15 to 30 min, 100 mm; gradient B, 0 to 5 min, 0 mm; 5 to 35 min, 0 to 430 mm; 35 to 40 min, 430 to 1000 mm; 40 to 45 min, 1000 mm; 45 to 60 min, 0 mm; and gradient C, 0 to 50 min, 0 to 450 mm; 50 to 55 min, 450 to 1000 mm; 55 to 70 min, 0 mm.

	RESULTS AND DISCUSSION

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Preparation of RG Substrates

The same sequential enzymic degradation procedure was performed on the hexasaccharide 1 (Fig. 1, peak a1) that was purified from an RG-hydrolase MHR-S digest (Mutter et al., 1994). The major product generated by -galactosidase treatment of 1 is the degalactosylated tetrasaccharide 3 (Fig. 1, peak b1). We assume that the minor components (Fig. 1, peaks b2 and b3) are partially degalactosylated oligosaccharides. Moreover, small amounts of Rha and GalA were released, which suggests that the -galactosidase fraction also contained rhamnohydrolase and galacturonohydrolase activities. The main product generated by treating the degalactosylated tetrasaccharide 3 with RG-rhamnohydrolase was the trisaccharide 5 (Fig. 1, peak c1). At the same time, some additional Gal was released (not shown), which could explain why peak b2 (a galactosylated fragment) was not detected anymore. We assume that peak b3, another presumptive galactosylated fragment, was also converted to the trisaccharide. The structure of peak c2 in Figure 1 is unknown. Nevertheless, we considered the oligosaccharide fraction to be suitable for use in investigating the mode of action of RG-galacturonohydrolase.

View larger version (26K): [in this window] [in a new window]   Figure 1. a, HPAEC of the purified hexasaccharide 1 fraction (peak a1; structures in Table I); b, this fraction after degalactosylation, which generates as major product tetrasaccharide 3 (peak b1); c, this fraction after degalactosylation and subsequent derhamnosylation, which generates as major product trisaccharide 5 (peak c1).

The substrate specificity of RG-galacturonohydrolase was further determined using apple MHR-S, a mixture of oligomers 7 to 10 (Table I) and the purified hexasaccharide 7 generated by RG-lyase treatment of MHR-S and subsequent SEC purification, purified linear RG oligomers 11 to 15 (Table I), and a mixture of RG oligomers with an average DP of 20 (16 in Table I; Renard et al., 1998).

Purification of RG-Galacturonohydrolase from A. aculeatus

View larger version (31K): [in this window] [in a new window]   Figure 2. Detailed purification scheme of RG-galacturonohydrolase from Pectinex Ultra SP produced by A. aculeatus.

Since other enzymes of interest were also present in the unbound fraction, purification was continued with this fraction, which was applied to an SP Sepharose cation exchanger at pH 4.25. RG-galacturonohydrolase activity eluted from the column at 124 mm NaCl. Pooled fractions were applied to a Q-Sepharose anion exchanger at pH 6.0. RG-galacturonohydrolase activity was not found in a distinct peak but was present in all three major protein peaks eluting from the column at 24, 35, and approximately 60 mm NaCl, in amounts that could be related to the protein content of the fractions. RG-rhamnohydrolase was also present in all major fractions but was most abundant in the 35 and 60 mm NaCl fractions. Many attempts were made to achieve separation of RG-galacturonohydrolase from RG-rhamnohydrolase. These included various different forms of SEC, ion-exchange chromatography, chromatofocusing, hydrophobic interaction chromatography, and affinity chromatography. None of these methods was effective. Finally, the best separation of the two enzymes was obtained using IMAC. The three fractions from the Q-Sepharose column were applied to a column of chelating Sepharose Fast Flow, which was loaded with CuCl_2. For the 35 mm Q-Sepharose fraction the elution pattern is shown in Figure 3. It can be seen that the majority of RG-rhamnohydrolase was not bound to the column, although a small amount eluted at the front of the major protein peak (at pH 5.56). RG-galacturonohydrolase eluted in the tail of the major protein peak. The unbound protein containing RG-rhamnohydrolase activity was pooled and used for de-rhamnosylation of substrates, as described above.

View larger version (19K): [in this window] [in a new window]   Figure 3. Chromatography of the protein fraction that was eluted from the Q-Sepharose column at 35 mm NaCl, on a chelating Sepharose Fast Flow column loaded with Cu2+ ions. For elution a pH gradient of pH 6.0 to 4.0 (buffer B) in 20 mm Bis-Tris containing 500 mm NaCl was used (Fig. 2). Solid line, A280; dotted line, percent buffer B; , RG-galacturonohydrolase activity; , RG-rhamnohydrolase activity (expressed as percentages of sugar released from the total amount present in the substrate).

Column fractions with the highest RG-galacturonohydrolase activity were pooled, and all resulting fractions formed were again applied to a chelating Sepharose column, this time using high-performance material to remove traces of RG-rhamnohydrolase. Finally, nine pools containing RG-galacturonohydrolase activity were obtained, in total representing approximately 0.01% (w/w) of the originally desalted protein of Pectinex Ultra SP. The proteins in all of these pools showed the same molecular mass upon SDS-PAGE. The purest fraction, based on side activity determination (see below), was chosen for characterization.

Characteristics of RG-Galacturonohydrolase

RG-galacturonohydrolase was tested toward various substrates to screen for other glycanase and/or glycosidase activities. HPSEC was used to detect a shift in molecular mass of the polymeric substrate, and HPAEC was used to detect whether sugar monomers or oligomers were released. No activity was found toward CM-cellulose, crystalline cellulose, xylan from oat spelts, soluble starch, potato arabino--(1,4)-galactan, larchwood arabino--(1,3)/(1,6)-galactan (stractan), linear arabinan, PGA with or without 1 mm CaCl₂ added, or pectin with a DM of 92.3. The same was true for pnp--l-Ara_f, pnp--d-Gal_p, pnp--d-Gal_p, pnp--d-Xyl_p, pnp--d-Xyl_p, pnp--d-Man_p, pnp--d-Man_p, pnp--l-Fuc_p, pnp--d-Fuc_p, pnp--d-Glc_p, pnp--d-Glc_p, pnp--l-Rha_p, pnp--d-Glc_pA, and pnp--d-Gal_pA.

The optimum pH of RG-galacturonohydrolase was found at pH 4.0 in McIlvaine buffers (Fig. 4a), and the enzyme was most stable between pH 2.5 and 7.0 for 1 h at 40°C and between pH 3.0 and 6.0 for 24 h at 40°C (Fig. 4b). The optimum temperature was 50°C in NaOAc buffer (pH 5.0; Fig. 4c), and RG-galacturonohydrolase was stable for at least 3 h at 60°C (Fig. 4d).

View larger version (28K): [in this window] [in a new window]   Figure 4. a, Optimum pH of RG-galacturonohydrolase, 100% = enzyme activity at optimum pH; b, pH stability of RG-galacturonohydrolase, 100% = activity of untreated enzyme; c, optimum temperature of RG-galacturonohydrolase, 100% = enzyme activity at optimum temperature; and d, temperature stability of RG-galacturonohydrolase, 100% = activity of untreated enzyme.

Mode of Action of RG-Galacturonohydrolase

View this table: [in this window] [in a new window]   Table II. Activity of RG-galacturonohydrolase toward structurally different RG fragments RG-galacturonohydrolase (0.42 µg mg1 substrate) was incubated with 0.025% (w/v) substrate solutions in 50 mm NaOAc buffer (pH 5.0), for 1 and 45 h at 40°C. Incubation mixtures and blanks were analyzed on HPAEC (gradient C). Explanation of symbols is given in Table I.

View larger version (18K): [in this window] [in a new window]   Figure 5. a, HPAEC of hexasaccharide 1 fraction (peak a1; structures in Table I) before (bottom) and after (top) 45 h of incubation with RG-galacturonohydrolase; b, tetrasaccharide 3 (peak b1) before (bottom) and after (top) 45 h of incubation with RG-galacturonohydrolase; c, trisaccharide 5 (peak c1) before (bottom) and after (top) 45 h of incubation with RG-galacturonohydrolase.

From Table II it is clear that the highest activity was obtained for trisaccharide 5, which contains a GalA at the nonreducing end in contrast to the two other oligomers 1 and 3. Figure 5c shows that peak c1, corresponding to trimer 5, is degraded under formation of GalA and a new peak, d1, which must be the dimer -Rha-(1,4)-GalA. The results therefore show that RG-galacturonohydrolase removes the GalA from the nonreducing end of RG fragments. The disaccharide -Rha-(1,4)-GalA, eluting at 13.5 min, is less retarded on the column than GalA, which can be attributed to the effect of Rha at the nonreducing end, as was already shown by Mutter et al. (1994). Peak d1 could be degraded completely into GalA and Rha upon subsequent degradation with RG-rhamnohydrolase (not shown). Only a trace of RG-galacturonohydrolase activity was found toward hexasaccharide 1, and Figure 5a shows that peak a1, corresponding to 1, was not degraded. Although 13% of the total GalA was released from tetrasaccharide 3, HPAEC (Fig. 5b) reveals that peak b1, corresponding to 3, was not degraded, and therefore the GalA released must be released from contaminating oligomers present in the fraction instead of from the reducing end of 3.

The partial ¹H-NMR spectra recorded just prior to and at intervals after the addition of RG-galacturonohydrolase to a mixture of RG oligosaccharides 13 and 14 (Table I), illustrating the stereochemical course of the reaction, are shown in Figure 6. During the first few minutes of the incubation a doublet at about 4.61 ppm (J 7.9 Hz), assigned to H-1 of GalA (Rees and Wight, 1971; Tjan et al., 1974) appeared and rapidly increased in intensity. Later in the incubation a small doublet at 5.30 ppm (J 3.8 Hz), due to H-1 of GalA (Rees and Wight, 1971; Tjan et al., 1974), became noticeable and almost certainly arose from the mutarotation of the initially formed -anomers. Other notable changes in the ¹H-NMR spectra during the incubation includes an increase in the resonance at about 5.21 ppm, assigned to H-1 of terminal nonreducing end -Rha residues, and a decrease in the resonance at about 5.25 ppm due to internal -Rha residues (Colquhoun et al., 1990; Schols et al., 1994). This confirms that the GalA is removed from the nonreducing end. Therefore, all the data clearly indicate that RG-galacturonohydrolase catalyzes the hydrolysis of -GalA-(1,2)--Rha linkages at the nonreducing end of the RG oligomers with inversion of anomeric configuration (e  a) and most likely operates via a single displacement reaction mechanism (Sinnott, 1990). This is similar to most other galacturonosyl hydrolases so far investigated (Biely et al., 1996; Pitson et al., 1998), although a digalacturonohydrolase (EC 3.2.1.82) from Selenomonas ruminantium was reported to catalyze glycosyl transfer (Heinrichová et al., 1992) and therefore probably acts with net retention of anomeric configuration.

View larger version (21K): [in this window] [in a new window]   Figure 6. Partial 1H-NMR spectra showing the stereochemical course of hydrolysis of linear RG oligomers by the RG-galacturonohydrolase. H-1 resonances of the GalA released are indicated (-GalA and -GalA).

Substrate Specificity of RG-Galacturonohydrolase

View this table: [in this window] [in a new window]   Table III. Activity of RG-galacturonohydrolase toward different GalA-containing substrates RG-galacturonohydrolase (0.76 µg µmol1 substrate) was incubated with substrate solutions, adjusted to approximately 150 µm nonreducing GalA residues in 50 mm NaOAc buffer (pH 5.0) for 1 and 45 h at 40°C. Incubation mixtures and blanks were analyzed on HPAEC using gradient A and C; polymeric substrates were also analyzed on HPSEC. Explanation of oligosaccharide codes is given in Table I.

RG-galacturonohydrolase was active toward all RG types of substrate, except for those with a us-GalA at the nonreducing end. The fact that RG-galacturonohydrolase is active toward MHR-S shows that some nonreducing GalA must be present in MHR-S, although only 0.4% of the total GalA present was released. The slight activity toward the mixture of oligosaccharides 7 to 10 (Table I) might be explained by the presence of contaminating larger original MHR-S fragments in the oligomer mixture containing GalA at the nonreducing end. From oligomers 11, 13, and 15 and mixture 16, essentially all available GalA was released. From the mixture of oligosaccharides 5 and 6 only 61% of all available GalA residues could be released instead of the expected 100%. This could be due to the fact that the degalactosylation was not complete in the batch used, and therefore not all material originally present was modified into products with a nonreducing GalA available, as was assumed in the calculation.

Although the RG-galacturonohydrolase activity toward pectin with a DM of 35 was only 1 unit mg¹, 13% of the available GalA residues could be released after 45 h. Van Rijssel et al. (1993) degraded citrus pectin (DM 62) with a PGA hydrolase from Clostridium thermosaccharolyticum and found that 5.7% (w/w) of this substrate could not be degraded by the enzyme. This so-called "limit pectin" was rich in GalA, Rha, Ara, and Gal (Rha:GalA = 0.54). To investigate the possibility that the released GalA from pectin with a DM of 35 and PGA in Table III originated from RG regions, the substrates were incubated with both RG-galacturonohydrolase and RG-rhamnohydrolase. In addition to GalA, Rha was also released (not shown), indicating that accessible RG regions were indeed present.

Kinetic Properties of RG-Galacturonohydrolase

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View this table: [in this window] [in a new window]   Table IV. Kinetic parameters for RG-galacturonohydrolase RG-galacturonohydrolase (5.2 ng mL1 incubation mixture) was incubated for 30 min at 40°C with six different substrate concentrations between 0.030 and 1.3 mm for oligosaccharides 11, 12, 14, and mixture 16 (structures in Table I) and between 0.83 and 5 mm for MHR-S, dissolved in 50 mm NaOAc buffer (pH 5.0). Incubation mixtures and blanks were analyzed using HPAEC (gradient A).

RG-galacturonohydrolase, in combination with RG-rhamnohydrolase, might be applied to remove RG fragments from acid-extracted HGs as used in industry, to improve the gelling properties. The combination of these RG exo-enzymes can also be used in the complete saccharification of biomass as for sugar beet pulp, from which process the resulting Rha monomers can be used as a precursor of aroma compounds such as furaneol (Micard et al., 1996). Finally, because they are capable of modifying RG structures, RG-galacturonohydrolase and RG-rhamnohydrolase might become important in the study of biologically active RGs such as sycamore RG I, which has been demonstrated to have wound-signal activity (Ryan et al., 1981). These exo-enzymes have not yet been found in plants. However, activity of another RG-specific enzyme, the RG-hydrolase, has recently been found in apples, grapes, and tomatoes (Gross et al., 1995).

	CONCLUSIONS

From the commercial enzyme mixture Pectinex Ultra SP, produced by A. aculeatus, an RG-galacturonohydrolase has been purified. This enzyme hydrolyzes the GalA residue from the nonreducing end of RG structures with inversion of anomeric configuration. To our knowledge, no such enzyme has been described in the literature. Being highly specific for RGs, and not active toward HGs, RG-galacturonohydrolase can be considered the latest in a series of RG-specific enzymes, after RG-hydrolase (rhamnogalacturonase, Schols et al., 1990), RG-acetylesterase (Searle-Van Leeuwen et al., 1992), RG-rhamnohydrolase (Mutter et al., 1994), and RG-lyase (Azadi et al., 1995; Mutter et al., 1996). Taking into account the substrate specificity and mode of action of the enzyme, the proposed systematic name is RG -d-galactopyranosyluronohydrolase.

	FOOTNOTES

   Received October 20, 1997; accepted January 29, 1998.

	ABBREVIATIONS

Abbreviations: DM, degree of methoxyation: no. of mol of methoxyl groups per 100 mol of GalA residues. DP, degree of polymerization. [E], enzyme concentration. GalA, d-galactopyranosyluronic acid. HG, homogalacturonan. HPAEC, high-performance anion-exchange chromatography. HPSEC, high-performance size-exclusion chromatography. IMAC, immobilized metal ion-affinity chromatography. k_cat, catalytic constant, k_cat/K_m is the specificity constant. MHR-S, saponified modified hairy regions of pectin. PAD, pulsed amperometric detection. PGA, polygalacturonic acid. pnp, p-nitrophenyl. R, -Rha (1,4)-linked to GalA, or Rha at the reducing end. RG, rhamnogalacturonan. RG-galacturonohydrolase, RG -d-galactopyranosyluronohydrolase. RG-hydrolase, RG -d-galactopyranosyluronide-(1,2)--l-rhamnopyranosyl hydrolase. RG-lyase, RG -l-rhamnopyranosyl-(1,4)--d-galactopyranosyluronide lyase. RG-rhamnohydrolase, RG -l-rhamnopyranosylhydrolase. Rha, l-rhamnopyranose. SEC, size-exclusion chromatography. us-GalA, --(4,5)-unsaturated GalA. V_max, maximum reaction rate.

	ACKNOWLEDGMENTS

Thanks are due to Ingeborg Boels and Simone Bouman for their valuable contribution in the purification and characterization of RG-galacturonohydrolase and to Dr. C.M.G.C. Renard (Institut National de la Recherche Agronomique, Nantes, France) for providing the linear RG oligomers.

	LITERATURE  CITED

Top Abstract Introduction Methods Results & Discussion References

Azadi P, O'Neill MA, Bergmann C, Darvill G, Albersheim P (1995) The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase. Glycobiology 5: 783-789 [Abstract/Free Full Text]

Beldman G, Van den Broek LAM, Schols HA, Searle-Van Leeuwen MJF, Van Laere KMJ, Voragen AGJ (1996) An exogalacturonase from Aspergillus aculeatus able to degrade xylogalacturonan. Biotechnol Lett 18: 707-712 [CrossRef]

Biely P, Benen J, Heinrichová K, Kester HCM, Visser J (1996) Inversion of configuration during hydrolysis of -1,4-galacturonidic linkage by three Aspergillus polygalacturonases. FEBS Lett 382: 249-255 [CrossRef][Medline]

Colquhoun IJ, De Ruiter GA, Schols HA, Voragen AGJ (1990) Identification by N.M.R. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with RGase. Carbohydr Res 206: 131-144 [Medline]

De Vries JA, Voragen AGJ, Rombouts FM, Pilnik W (1981) Extraction and purification of pectins from alcohol insoluble solids from ripe and unripe apples. Carbohydr Polym 1: 117-127

Fersht A (1985) Enzyme Structure and Mechanism. WH Freeman and Co., New York

Gross KC, Starrett DA, Chen H-J (1995) Rhamnogalacturonase, -galactosidase, and -galactosidase: potential roles in fruit softening. Acta Hortic 398: 121-129

Heinrichová K, Dzúrová M, Rexová-Benková L (1992) Mechanism of action of d-galacturonan digalacturonohydrolase of Selenomonas ruminantium on oligogalactosiduronic acids. Carbohydr Res 235: 269-280 [Medline]

Labavitch JM, Freeman LE, Albersheim P (1976) Structure of plant cell walls. Purification and characterization of a -(1,4)-galactanase which degrades a structural component of the primary cell wall of dicots. J Biol Chem 251: 5904-5910 [Abstract/Free Full Text]

Micard V, Renard CMGC, Thibault J-F (1996) Enzymatic saccharification of sugar-beet pulp. Enzyme Microb Technol 19: 162-170 [CrossRef]

Mutter M, Beldman G, Schols HA, Voragen AGJ (1994) Rhamnogalacturonan -l-rhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin. Plant Physiol 106: 241-250 [Abstract]

Mutter M, Colquhoun IJ, Beldman G, Schols HA, Bakx EJ, Voragen AGJ (1998) Characterization of recombinant rhamnogalacturonan -l-rhamnopyranosyl-(1,4)--d-galactopyranosyl uronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturaonan I regions of pectin. Plant Physiol 117: 141-152 [Abstract/Free Full Text]

Mutter M, Colquhoun IJ, Schols HA, Beldman G, Voragen AGJ (1996) Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan -l-rhamnopyranosyl-(1,4)--d-galactopyranosyluronide lyase. Plant Physiol 110: 73-77 [Abstract]

Pilnik W, Voragen AGJ (1993) Pectic enzymes in fruit and vegetable juice manufacture. In T Nagodawithana, G Reed, eds, Enzymes in Food Processing. Academic Press, London, pp 363-399

Pitson SM, Mutter M, Van den Broek LAM, Voragen AGJ, Beldman G (1998) Stereochemical course of hydrolysis catalyzed by -l-rhamnosyl and -d-galacturonosyl hydrolases from Aspergillus aculeatus. Biochem Biophys Res Commun 242: 552-559 [CrossRef][Medline]

Pitson SM, Voragen AGJ, Beldman G (1996) Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases. FEBS Lett 398: 7-11 [CrossRef][ISI][Medline]

Rees DA, Wight AW (1971) Polysaccharide conformation for -1,4-galacturonan and the kinking function of l-rhamnose residues in pectic substances. J Chem Soc B: 1366-1372

Renard CMGC, Lahaye M, Mutter M, Voragen AGJ, Thibault J-F (1998) Isolation and structural characterisation of rhamnogalacturonan oligomers prepared by controlled acid hydrolysis of sugar-beet pulp. Carbohydr Res (in press)

Renard CMGC, Thibault J-F, Mutter M, Schols HA, Voragen AGJ (1995) Some preliminary results on the action of rhamnogalacturonase on rhamnogalacturonan oligosaccharides from beet pulp. Int J Biol Macromol 17: 333-336 [Medline]

Rombouts FM, Spaansen CH, Visser J, Pilnik W (1978) Purification and some characteristics of pectate lyase from Pseudomonas fluorescens GK-5. J Food Biochem 2: 1-22

Ryan CA, Bishop P, Pearce G, Darvill AG, McNeil M, Albersheim P (1981) A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities. Plant Physiol 68: 616-618 [Abstract/Free Full Text]

Schols HA, Geraeds CCJM, Searle-Van Leeuwen MJF, Kormelink FJM, Voragen AGJ (1990) Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr Res 206: 105-115 [CrossRef]

Schols HA, Vierhuis E, Bakx EJ, Voragen AGJ (1995) Different populations of pectic hairy regions occur in apple cell walls. Carbohydr Res 275: 343-360 [CrossRef][ISI][Medline]

Schols HA, Voragen AGJ, Colquhoun IJ (1994) Isolation and characterization of rhamnogalacturonan-oligomers, liberated during degradation of pectic hairy regions by RGase. Carbohydr Res 256: 97-111 [CrossRef][Medline]

Searle-Van Leeuwen MJF, Van den Broek, LAM, Schols HA, Beldman G, Voragen AGJ (1992) Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins. Appl Microbiol Biotechnol 38: 347-349

Sinnott ML (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev 90: 1171-1202 [CrossRef][ISI]

Thibault J-F, Renard CMGC, Axelos MAV, Roger P, Crépeau M-J (1993) Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr Res 238: 271-286 [CrossRef]

Tjan SB, Voragen AGJ, Pilnik W (1974) Analysis of some partly and fully esterified oligogalacturonic acids by P.M.R. spectrometry at 220 MHz. Carbohydr Res 34: 15-32 [CrossRef]

Van Cutsem P, Messiaen J (1994) Biological effects of pectic fragments in plant cells. Acta Bot Neerl 43: 231-245

Van Deventer-Schriemer WH, Pilnik W (1976) Fractionation of pectins in relation to their degree of esterification. Lebensm Wiss Technol 9: 42-44

Van Deventer-Schriemer WH, Pilnik W (1987) Studies on pectin degradation. Acta Alimentaria 16: 143-153

Van de Vis JW (1994) Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans. PhD thesis. Koninklijke Bibliotheek, The Hague, pp 89-108

Van Rijssel M, Gerwig GJ, Hansen TA (1993) Isolation and characterization of an extracellular glycosylated protein complex of Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity. Appl Environ Microbiol 59: 828-836 [Abstract/Free Full Text]

Voragen AGJ (1972) Characterization of pectin lyases on pectins and methyl oligogalacturonates. Agric Res Rep 780: 1-121

Voragen AGJ, Schols HA, Gruppen H (1993) Structural studies of plant cell-wall polysaccharides using enzymes. In F Meuser, DJ Manners, W Siebel, eds, The Proceedings of the International Symposium on Plant Polymeric Carbohydrates, Berlin, July 1-3 1992. The Royal Society of Chemistry, Cambridge, UK, pp 1-15

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