Penicillium daleae, a soil fungus able to degrade rhamnogalacturonan II, a complex pectic polysaccharide

Penicillium daleae, a soil fungus able to degrade rhamnogalacturonan II, a complex pectic polysaccharide Ste´phane Vidal,* Jean-Michel Salmon,† Pascale Williams,* and Patrice Pellerin* *Unite´ de Recherches des Polyme`res et des Techniques Physico-Chimiques, †Unite´ de Recherches de Microbiologie et de Technologie des Fermentations, Institut National de la Recherche Agronomique, Institut des Produits de la Vigne, Montpellier Cedex, France A strain of Penicillium daleae has been isolated from a forest soil sample for its ability to degrade monomeric rhamnogalacturonan-II (mRG-II), a complex pectic polysaccharide ubiquitous in the primary plant cell wall. Monomeric RG-II, a most unusual polysaccharide in terms of composition and structure, is resistant to all known pectinolytic enzymes used in the fruit- and vegetable-processing industry. P. daleae has been cultured in a minimum mineral medium supplemented with 0.5% mRG-II as the sole carbon source. The degradation of the substrate in the culture supernatant was followed by high performance size-exclusion chromatography. P. daleae growth was supported by the degradation of 75% of the initial mRG-II. Monomeric RG-II degradation was followed by gas chromatography-mass spectrometry of the alditol acetates and trimethylsilylated derivatives of the constitutive monosaccharides at different times in the culturing process. Sequential mRG-II degradation led to a resistant core after 31 days of culture, representing 25% of the initial molecule which has been characterized. The degradation of mRG-II indicated that P. daleae is a potential source of new pectinases whose mode of action requires further elucidation. Such enzymes seemed to be exposed to the surface of fungal cell walls or were present in the periplasmic compartment. © 1999 Elsevier Science Inc. Keywords: Rhamnogalacturonan II; pectic polysaccharide; Penicillium daleae; plant cell wall; pectinase

Introduction Rhamnogalacturonan II (RG-II) is a low molecular weight, complex pectic polysaccharide1 which was first isolated from suspension-cultured sycamore cells (Acer pseudoplatanus).2 RG-II has been characterized in a wide variety of plant species from Pteridophytes to Spermatophytes, and also in several organs including fruits from kiwi (Actinidia deliciosa),3 grape (Vitis vinifera),4 apple (Malus domestica), and tomato (Solanum lycopersicum);5 roots from sugar-beet (Beta vulgaris),6 radish (Raphanus sativus),7 and carrot (Daucus carota),5 leaves from Panax ginseng,8 Arabidopsis thaliana and Polypodium vulgare,1 and pea (Pisum sativum)

Address reprint requests to Dr. P. Pellerin, Institut National de la Recherche Agronomique, Institut des Produits de la Vigne, Unite´ de Recherches des Polyme`res et des Techniques Physico-Chimiques, 2 place Viala, F-34060 Montpellier, France Ste´phane Vidal is affiliated with Gist-Brocades France S.A., 15 rue des Comtesses, BP 239, Seclin, F-59472, France Received 23 April 1998; revised 3 September 1998; accepted 18 September 1998

Enzyme and Microbial Technology 24:283–290, 1999 © 1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

stems.10 In a recent study, the presence of RG-II has been detected in 24 plant species11 confirming its ubiquity. RG-II accounts for 1–5% of growing plant cell walls in dicots and nongraminaceous monocots and has also been detected in graminaceous cell walls12 with a much lower content. The complexity of RG-II is emphasized by the accepted model (Figure 1) for its organization which is based on a backbone of at least eight 1,4-linked a-d-galacturonosyl residues carrying four oligosaccharide side chains.13 The degree of polymerization is about 30 with 12 different sugars including unusual monosaccharides such as apiose, 2-O-methyl-l-fucose, 2-O-methyl-d-xylose, Dha (3-deoxyd-lyxo-heptulosaric acid),14 Kdo (3-deoxy-d-manno-octulosonic acid),15 and aceric acid (3-C-carboxy-5-deoxy-lxylose),16 the only branched acidic deoxy-sugar described in the plant kingdom. Furthermore, several monosaccharides which are more common in other cell wall polysaccharides are involved in peculiar linkages as fully substituted rhamnosyl, 2,4- and 3,4-linked galacturonosyl or 3,4-linked fucosyl residues. RG-II has been shown to have the same structure in every plant from which it has been isolated, with minor differences in the length of the homogalacturonan

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Figure 1 Hypothetical structure of mRG-II. Glycosyl residues of side chains A to D are numbered according to the model sequence. The attachment sites of side chains on the homogalacturonan backbone are not yet determined. The hypothetical structure of the resistant core of mRG-II left in the supernatant of a 31-day-old culture of P. daleae is surrounded by a dotted line

backbone or in the nature of the nonreducing terminal residues on the side chains.8,17,18 Recent studies have shown that RG-II is present in cell walls predominantly as a dimer formed by the cross-linkage of two molecules of RG-II by a 1:2 borate-diol ester located on one apiosyl residue.10,11,19 This result represented the first localization of the “nonavailable” boron present in cell walls. Boron is an essential micronutrient for dicots and monocots; a boron deficiency results in a disorganization of cell expansion generating swollen abnormal cells. The quantitative association between boron and RG-II in at least 15 plant species11 provides a compelling argument that RG-II plays a major role in the formation of a cross-linked pectic matrix essential for the structure and functions of the walls of growing plants.20 This structural role gave the first explanation for the highly conserved structure of RG-II with regard to the biosynthetic cost it might represent for plants. The structural model given in Figure 1 has been established with RG-II released from sycamore cell walls treated with a purified fungal endo-a-1,4-polygalacturonase which degraded the homogalacturonan chains of native pectins.2 The structure of RG-II isolated from a commercial enzyme preparation,14 wines,4,17 or fruit juices5 obtained with liquefying enzyme preparation (Pectinex Ultra SP-L and Rapidase Liq) was indistinguishable; therefore, RG-II can be defined as an enzyme-resistant pectic fragment.5 This resistance to known pectolytic enzymes may be explained by the predominance of unusual linkages, anomeries or sugar residues in the molecule, combined to a steric hin284

drance due to its high level of ramification; however, the abundance and ubiquity of RG-II indicated that the corresponding hydrolytic enzymes should be present in natural ecosystems such as soils or composts. The resistance of RG-II to the enzymes produced by fruits, Aspergilli spp, and Saccharomyces cerevisae makes it a predominant wine and fruit-juice polysaccharide.4,5 RG-II in wine has been reported to be involved in the fouling of filtration membranes,21 in the prevention of potassium hydrogen tartrate crystallization,22 and in the complexation of lead.23 Solutions to these problems may be obtained by the use of new pectinases specific to RG-II hydrolysis. Penicillium spp are known to be pectinolytic fungi abundant in soils. Penicillium daleae, belonging to the Penicillium nigricans series, has been isolated from both forest and meadow soils and described for its ability to grow by degrading pectins.24 This study deals with the isolation of a strain of P. daleae remarkable for its ability to degrade RG-II and with preliminary studies on the degradation pathway of RG-II and on the localization of the corresponding enzymes.

Materials and methods Isolation of wine rhamnogalacturonan II Total polysaccharides were precipitated by addition of five volumes of cold ethanol on the ultrafiltration retentate (molecular

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Degradation of rhamnogalacturonan II: S. Vidal et al. weight cut-off, 20 kDa) from 600 l of a red wine as described previously.25 Total polysaccharides were chromatographed on an anion-exchange DEAE-Macroprep column (5 3 80 cm; Bio-Rad, Richmond, CA) equilibrated in 40 mm sodium citrate buffer pH 4.6. The adsorbed polysaccharides were separated by stepwise elution with NaCl in starting buffer. Four RG-II-containing fractions were obtained. Yeast mannoproteins were eliminated by affinity chromatography on a Concanavalin A-Ultrogel column (5 3 70 cm; Biosepra, Villeneuve la Garenne, France) equilibrated in 50 mm sodium acetate buffer pH 5.6 containing 150 mm NaCl, 1 mm CaCl2, 1 mm MnCl2, and 1 mm MgCl2. RG-II was then separated from high molecular weight arabinogalactan-proteins by size-exclusion chromatography on a Sephacryl S-400 HR column (5 3 80 cm; Pharmacia, Uppsala, Sweden) equilibrated in 50 mm sodium acetate buffer pH 5 containing 50 mm NaCl. Finally, the RG-II preparations were dialyzed (molecular weight cut-off 8 –10 kDa) against water and freeze-dried.

Characterization of monomeric RG-II A 13 g preparation of RG-II was used as the substrate for the screening and isolation of the fungal strains as well as for the study of the degradation pathway. This preparation, previously reported as RG-II2,17 contained about 98% of monomeric RG-II (mRG-II) as indicated by high resolution size-exclusion chromatography on a Superdex 75-HR column (1 3 30 cm; Pharmacia) equilibrated at 0.6 ml min21 in 25 mm ammonium formate pH 5.2. The elution was monitored by refractometry. The elution profile17 indicated that the preparation is devoid of any oligosaccharide contaminant. Glycosyl-residue and glycosyl-linkage analyses17 indicated that this purified mRG-II corresponded to the accepted model given in Figure 1 with a backbone of eight a-1,4-galacturonosyl residues; three were methyl esterified. Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) performed on a Hewlett-Packard LDI 1700XP mass spectrometer,17 showed that mRG-II had a molecular weight of approximately 4,720 in accordance with the postulated model.

Culture conditions All the cultures were incubated aerobically at 25°C under static conditions. Liquid medium. The liquid medium contained in 1 l of mineral medium (NH4NO3, 2 g; K2HPO4, 1 g; MgSO4 z 7H2O, 0.5 g; KCl, 0.5 g; FeSO4, 10 mg); 1 ml of Heller solution (1 mg ZnSO4, 0.1 mg MnSO4 z H2O, 0.03 mg CuSO4 z 5H2O, 0.03 mg AlCl3, 0.03 mg NiCl2 z 6H2O, 0.01 mg KI, and 1 mg boric acid); 1 ml of vitamin solution (B1 and H vitamins each at 80 mg l21), 2 ml of streptomycin solution at 50 mg ml21, 2 ml of tetracyclin solution at 5 mg ml21 and 10 ml of penicillin G solution at 10,000 U ml21. Five ml of this mineral medium was supplemented with 0.5% mRG-II and/or with 2% citrus pectin, apple pectin, and glucose. Solid medium. The strain of P. daleae was isolated on solid mineral medium supplemented with 2% agar and 0.5% mRG-II in Petri dishes. Inoculation. The different cultures were inoculated with mycelial disks cut from the margin of the colonies obtained on the solid medium, or from 1–5% (fresh weight) of the total mycelium obtained in liquid cultures.

Screening and isolation of microorganisms Samples originating from various ecosystems were tested for the presence of microorganisms able to grow on 0.5% mRG-II as a

unique carbon source. The liquid medium was inoculated with 2% of the different samples tested: winery subproduct composts, soils and wastewater treatment sludges. The recovered microorganisms were spread on plates containing the solid medium. The microorganisms growing on these plates were subsequently tested for their capability to degrade mRG-II in liquid medium. The selected strains were sent for identification to Professor Roquebert, Museum National d’Histoire Naturelle (Paris).

Degradation monitoring The degradation of mRG-II in the liquid cultures was followed by high performance size-exclusion chromatography (HPSEC) on two serial Shodex OH-Pak KB-803 and KB-805 columns (Showa Denko; Tokyo, Japan) equipped with a KB-800 guard column and equilibrated at 1 ml min21 in 0.1 m LiNO3. Samples (25 ml) of the culture supernatants were taken out initially and at different times during fungal growth, centrifuged (2 min at 12,000 rpm), and injected on the HPSEC columns. The elution of mRG-II and its degraded products was monitored on a refractometer detector (Erma ERC-7512, Tokyo, Japan).

Analytical methods At different times during the culturing process, P. daleae mycelium grown on mRG-II was separated from the culture supernatant by filtration. Quantification of growth. At each culture time, the fungal growth was evaluated by determining both the dry weight of the total mycelium recovered from cultures and its corresponding chitin content. Samples of freeze-dried mycelium (0.5 mg) were hydrolyzed by 6 m HCl (16 h at 80°C), and the released glucosamine was quantified by high performance anion-exchange chromatography (HPAEC) on a Dionex system equipped with a Pulsed Amperometric Detector, a CarboPac PA-1 column (0.4 3 25 cm; Dionex, Sunnyvale, CA) and a CarboPac PA-1 guard column (0.4 3 5 cm). The elution at 1 ml min21 was performed under isocratic conditions at 14 mm NaOH and the detection was optimized with a post-column addition of 300 mm NaOH at 0.3 ml min21. Fucose was used as internal standard. Glycosyl-residue composition. Residual polysaccharides in the culture supernatant were recovered after dialysis and freezedrying. Their glycosyl-residue compositions were determined by gas chromatography (GC) analysis of the alditol acetates26 and of the per-O-trimethylsilylated methyl glycosides,17 separated on fused silica DB-225 (170°C) and DB-1 (temperature programming at 120°C to 200°C at 1.5°C min21) capillary columns (30 m 3 0.32 mm i.d., 0.25 mm film) with H2 as the carrier gas, respectively. The identity of each peak was confirmed by chemical ionization-mass spectrometry (CI-MS) on a HP-5989 MS-engine (Hewlett-Packard, Avondale, PA). Glycosyl-linkage determination. Glycosyl-linkages were determined by GC– electron impact-mass spectrometry of the partially methylated carboxyl-reduced alditol acetates15,17 analyzed on both fused-silica DB-225 (temperature programming 170°C for 15 min and then 5° min21 to 210°C) and DB-1 (temperature programming 145°C for 10 min and then 2° min21 to 190°C) columns. Analysis of mRG-II backbone fragments. Homogalacturonan backbone fragments were obtained by partial acid hydrolysis using 0.1 m trifluoroacetic acid for 16 h at 80°C and analysis with HPAEC on the same Dionex system.17,18 The elution at 1 ml min21 was performed with a gradient of sodium acetate in 100 mm NaOH: 100 mm sodium acetate (0 –5 min); a linear gradient up to

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Papers 500 mm sodium acetate (5–25 min); a linear gradient up to 700 mm sodium acetate (25–50 min). The column was reequilibrated under initial conditions for 10 min prior to sample injection. The degrees of polymerization (dp) of the homogalacturonan fragments were directly determined by comparison with the elution times of 1,4-linked a-d-oligogalacturonides generated by treating a solution of polygalacturonic acid at 0.2% in 0.1 m sodium acetate pH 4.8 with a purified endopolygalacturonase (Megazyme, Australia, 4 nkat ml21, 40°C, 16 h).

Location of enzymes having mRG-II degrading activity To locate the enzymes responsible for the mRG-II degrading activity, a culture was grown at 25°C on 2.5 mg ml21 mRG-II. After 15 days of cultivation, the degradation of mRG-II was checked by HPSEC as previously described. Mycelium was separated from the culture supernatant by filtration on a 0.6 mm nylon mesh. Incubation of mRG-II with culture supernatant. To detect the presence of mRG-II degrading enzymes in the P. daleae culture supernatant, mRG-II was added at the final concentration of 2.5 mg ml21 to 1 ml of the crude or tenfold concentrated (Centricon 30; cut-off 30 kDa; Amicon, Beverly, MA) culture supernatant and the incubation was performed at 25°C for 24 h. The influence of the addition of 1 mm dithiothreitol (DTT) to the concentrated supernatant was also tested. Cell-free extract preparation. In parallel to the incubations with the culture supernatant, 50 –200 mg (fresh weight) of P. daleae mycelium grown on mRG-II were mixed with 0.45 g glass beads of 0.45– 0.5 mm diameter) in 250 ml of morphilino-ethane-sulfonic acid (MES) buffer at 50 mm pH 6 containing 1 mm DTT and 1 mm phenylmethylsulfonylfluoride.27 The mixture was crushed on a MM 2000 Retsch apparatus (Bioblock, Illkirch, France) for 5 min at 4°C, and centrifuged at 3,000 g for 5 min). Monomeric RG-II was added to the resulting supernatant at the final concentration of 2.5 mg ml21 and incubated overnight at 25°C.

Results and discussion Screening of microorganisms able to degrade mRG-II The liquid medium used for the screening and culture of fungi able to grow on mRG-II as a source of carbon and energy was inoculated with 2% of the different samples tested: winery subproduct composts, soils, and wastewater treatment sludges. A significant fungal growth was only observed in the culture flasks inoculated with the soil samples; however, bacteria (cocci and bacilli) were observed in most samples when no antibiotics were added to the culture medium. Their growth could be inhibited by the combined addition of three antibiotics (penicillin G, tetracyclin, and streptomycin). The mycelia produced on mRG-II were collected, suspended in fresh liquid medium, and spread on plates containing the same medium but complemented with 2% agar. Different strains were isolated from the margin of the distinct colonies obtained after 7– 8 days of culture at 25°C, and subsequently tested for their capability to degrade mRG-II when cultivated again in the same liquid medium. A Penicillium strain obtained from the soil of a mixed 286

Figure 2 Monitoring of mRG-II degradation in the culture supernatant of P. daleae. Aliquots (25 ml) of culture supernatant, removed initially (a) and after 8 days of culture (b), were analyzed by HPSEC on two serial Shodex OH-Pak KB-803 and KB-805 columns. The elution of polysaccharides was monitored with a differential refractometer

maritime pine and birch-tree forest was selected since its growth was reproducible and associated unambiguously with the degradation of mRG-II in the medium. This strain has been identified as Penicillium daleae Zaleski by Professor Roquebert (Museum National d’Histoire Naturelle, Paris) and deposited at the Collection Nationale des Cultures de Micro-Organismes (Institut Pasteur, Paris) with the reference I-1577. P. daleae is a rather uncommon fungus, generally isolated from forest soils and already known for its ability to degrade pectins and starch;24 however, its growth on rhamnogalacturonan II had not been reported previously.

Culture conditions of P. daleae Monitoring of mRG-II degradation. The isolated P. daleae strain was cultivated under static conditions in aerobiosis at 25°C in the liquid mineral medium containing 2.5 mg ml21 mRG-II as the sole carbon source. The mRG-II preparation was devoid of any oligosaccharide, but several components of the culture medium were eluted as minor peaks between 19.5–21 min (Figure 2). After 5 days of culture, fungal growth was observed as a white mycelium spread on the air-liquid interface of inoculated tubes. Sporulation then began and induced the appearance of a green-brown color. After 8 days of culture, an aliquot of culture supernatant was removed and analyzed by HPSEC. Comparison of the HPSEC profile obtained to that of a 2.5 mg ml21 native mRG-II solution indicated a 45% decrease in the mRG-II peak area with a shift (0.1 min) in its elution time (Figure 2) while no modification of the profile could be observed after 18.5 min; therefore, the growth of P. daleae had been supported by the sole degradation of mRG-II in the liquid medium.

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Degradation of rhamnogalacturonan II: S. Vidal et al.

Figure 3 HPSEC analysis of polysaccharides in the culture supernatant of P. daleae, cultivated in the presence of both mRG-II (elution time 17–18 min) and apple pectin (elution time 13–18 min). Aliquots (25 ml) were removed initially (a) and after 21 days (b) of culture and analyzed on two serial Shodex columns

Influence of other carbohydrate sources. The influence of several carbohydrates on the growth of P. daleae and on the degradation of mRG-II was tested. The strain of P. daleae was cultivated in the presence of mRG-II alone, or in addition to other carbon sources (e.g., glucose, cellulose, pectins). In the case of cultures with mRG-II plus other carbohydrates, mRG-II was not degraded after 3 weeks of cultivation while the additional carbon source supported the fungal growth (Figure 3). This preliminary study indicated that mRG-II is used for the growth of P. daleae only when other carbon and energy sources are not available.

Preliminary study of the pathway of mRG-II degradation In order to study in parallel the fungal growth, the degradation rate, and the degradation pathway of mRG-II, seven culture tubes containing 25 mg of mRG-II in 5 ml of liquid medium were inoculated simultaneously and under the same conditions with P. daleae. The contents of one tube were harvested at a different time of culture; the mycelium was separated from the culture supernatant by filtration. The different times the culture was assayed were: initial, 4, 7, 11, 18, 21, and 31 days. The recovered mycelium was freeze-dried directly whereas the supernatant was dialyzed (molecular weight cut-off 1 kDa) before freeze-drying to recover the residual core of mRG-II. Fungal growth. P. daleae growth was followed by the determination of both the dry weight of the mycelium recovered at each culture time and its respective chitin content (Figure 4a). The fungal growth on mRG-II occurred very slowly after a 2-day lag phase. During this growth, the observed increase in mycelium dry weight was well correlated to the whole biomass chitin content, a characteristic component of ascomycete cell walls. This slow fungal growth reached a plateau after 18 days of culture. An

Figure 4 Growth of P. daleae and monitoring of mRG-II degradation. The growth of the fungus (a) in 5 ml of culture medium containing 5 mg ml21 of mRG-II was followed as the freeze-dried mycelium weight (}) or as its chitin content measured as the glucosamine released by acid hydrolysis (Œ). The degradation of mRG-II (b) was followed as the decrease in the dry weight of residual polysaccharides (}) in each culture supernatant (5 ml), or as the decrease in their corresponding area (Œ) by HPSEC analysis

increase in the chitin content of the mycelium was observed (from 1% after 4 days to 8% after 17 days of culture). Such a chitin accumulation in the fungal cell wall is known to occur when fungi are cultured under limiting conditions.28,29 mRG-II degradation monitoring. The degradation of mRG-II was followed at each culture time by HPSEC analysis of the culture supernatant corrected for evaporation. Peak area and elution time of the residual polysaccharides (elution range 17–18.5 min) present in the culture supernatant were followed in comparison with those of a native 5 mg ml21 mRG-II solution. The degradation rate was calculated as a percentage of the peak area of the native mRG-II solution. The values obtained were in perfect accordance with the decrease in dry weight of the residual polysaccharides obtained after dialysis (molecular weight 1 kDa) and freeze-drying of the culture supernatant (Figure 4b). When the culture was stopped, a resistant core, representing approximately 25% of the initial mRG-II, was still present in the culture medium (Figure 5). During fungal growth, no newly released oligosaccharide could be detected in the culture supernatant since the area of peaks eluted between 18.5 and 21 min remained constant. The

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Papers Table 1 Glycosyl-residue composition and degree of polymerization of the homogalacturonan backbone of the residual polysaccharides in the supernatant at different times of culture Glycosyl residuea

Initial

7 days

11 days

18 days

31 days

Api Ara Rha Fuc 2-O -Me-Xyl 2-O -Me-Fuc Aceric acid Gal Gal A Glc A Dha Kdo dpb

2.0 3.6 5.1 1.0 1.0 1.0 0.9 3.1 10.9 0.8 0.9 1.0 8–9

1.9 3.1 3.1 0.5 0.5 1.0 0.8 2.1 7.3 0.4 1.6 0.9 7–8

2.7 2.5 2.9 0.2 0.2 1.0 0.9 1.8 7.2 0.4 0.6 1.1 6–7

1.0 1.9 2.2 0.1 0.1 1.0 0.4 2.3 5.9 0.2 1.3 1.3 4–5

1.1 0.3 1.0 0.0 0.0 1.0 1.0 0.9 2.5 0.1 0.5 0.8 3–4

a Relative molar ratios determined by GC analysis of the per-O trimethylsilylated methyl glycosides expressed on the basis of one 2-O -Me-fucosyl residue b Degree of polymerization of the homogalacturonan backbone determined by HPAEC analysis

Figure 5 HPSEC elution profiles of native mRG-II (a) and residual polysaccharides present in P. daleae culture supernatant after 4 (b), 7 (c), 11 (d), or 21 (e) days of culture

degraded part of mRG-II represented 75% of the initial molecule, and the corresponding increase in total fungal biomass reached only 0.8 mg ml21. Such a discrepancy indicated that only a part of the mRG-II molecule degraded is assimilated by P. daleae. As a consequence, the biomass yield from mRG-II was low even after 15 days of culture. Due to its complex structure and composition, especially with the presence of several unusual sugars, mRG-II appears as a rather bad substrate for P. daleae.

Analysis of mRG-II degradation Glycosyl-residue compositions of the residual polysaccharides, recovered from the dialyzed culture supernatant at the different times indicated above, have been compared to that of the native mRG-II preparation. To facilitate the explanation of the degradation pathway, sugar residues have been numbered17 according to the model sequence of the oligosaccharide side chains A to D.30 Evolution of glycosyl-residue composition. The interpretation of the glycosyl-residue composition analyses (Table 1) to follow the degradation of the mRG-II molecule during the culture is facilitated by the presence of several sugars specific of a side chain of the molecule: 2-O-Me-xylose, glucuronic acid, and fucose are present only in side chain A; 2-O-Me-fucose and aceric acid in side chain B; Kdo and Dha the markers of side chains C and D, respectively; however, several sugars belong to different chains. Apiosyl residues are distributed among side chains A and B, rhamnosyl residues among side chains A, B, and C, and 288

galacturonosyl residues among the backbone and side chain A. GC-MS analyses of the alditol acetate derivatives obtained with the native and degraded forms of mRG-II indicated that the total amount of 2-O-Me-fucose (residue B49) was constant during the culture. This result revealed that 2-O-Me-fucose was neither metabolized by the fungus nor released from the undegraded fragments even in a 31-day-old culture; thus, glycosyl-residue compositions have been expressed as relative molar ratios calculated on the basis of one residue of 2-O-Me-fucose. Analyses of TMS derivatives allowed quantification of neutral and acidic sugar residues at the same time; the values obtained for neutral sugars are in accordance with the alditol acetate derivative analyses. In the same way, the molar ratios calculated for galacturonosyl residues were in accordance with the dp of the homogalacturonan backbone fragments released by partial acid hydrolysis. The evolution of relative molar ratios during the culture indicated that 2-O-Me-xylose (A39), fucose (A3), and glucuronic acid (A4) were lost simultaneously. Their content decreased by 50% after 7 days of culture, and 90% after 18 days, and they were absent in the final resistant polysaccharide after 31 days of cultivation. The simultaneous loss of one residue of galactose was also observed whereas one apiosyl residue was lost in a later phase of the culture. Nonreducing terminal arabinosyl and rhamnosyl residues of side chains B, C, and D also decreased dramatically. The decrease in the galacturonic acid content could be attributed both to the loss of nonreducing terminal galacturonosyl residues of side chain A (A29 and A20) and to a partial degradation of the homogalacturonan backbone whose average dp reduced from 9 to less than 4. On the contrary, the relative molar ratios of aceric acid (B3) and Kdo were remarkably stable. This preliminary study confirmed that only a specific part of the mRG-II molecule is released by the hydrolytic enzymes produced by P. daleae. The ob-

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Degradation of rhamnogalacturonan II: S. Vidal et al. tained results pointed to the quantitative loss of side chain A, and the decrease by half of the average dp of the homogalacturonan backbone. The other side chains have been degraded to a lesser extent since only terminal nonreducing residues have been released. Resistant core. As mentioned previously, the specific degradation of 75% of the mRG-II molecule led to the presence of a residual resistant polysaccharide in the supernatant after 31 days of culture. Glycosyl-residue analyses and a preliminary determination of its glycosyl-linkage composition (data not shown) indicated that the residual fragment proceeding from mRG-II degradation (Figure 1) is based on a short homogalacturonan backbone (dp 3– 4) which carries Kdo and a shortened side chain B (up to residue B49); however, the resistant polysaccharides in the final culture supernatant are still an heterogeneous mixture since arabinose and Dha are present as nonstoichiometric residues. The purification and characterization of these resistant fragments will allow improved knowledge of the fine structure mRG-II by determining the order in which the side chains are attached to the homogalacturonan backbone.

Location of the mRG-II degrading activities Fungal pectinolytic enzymes are generally reported to be excreted in the culture medium. The culture supernatant, obtained after removing the mycelium from a two-week-old culture by filtration, was assayed for the presence of mRG-II degrading enzymes. Monomeric RG-II was incubated with crude or ten-times concentrated cellfree culture supernatant. After 48 h of incubation, the HPSEC elution profiles obtained were indistinguishable from that of a native mRG-II solution, indicating that the enzymes responsible for the degradation of mRG-II could not be detected in the culture supernatant. The addition of 1 mm DTT did not modify this result. Monomeric RG-II was also incubated with the cellfree extract obtained by crushing the whole mycelium recovered from the same 2-week-old culture. After 48 h of incubation at 25°C, a degradation of about 50% of the initial substrate could be observed by HPSEC, indicating that the enzymes responsible for partial hydrolysis of mRG-II were released from the mycelium. These enzymes might be either located in the cytoplasmic compartment or associated to the cell wall; however, the presence of the degraded mRG-II fragments in the culture supernatant throughout the culture, and the molecular weight of mRG-II which is apparently inconsistent with a specific uptake through the plasma membrane, are compelling arguments to assume that the enzymes are exposed to the surface of fungal cell walls or are present in the periplasmic compartment.

enzymes may also be used in the wine and fruit-juice industry for resolving several problems such as the fouling of microfiltration membranes, and prevention of potassium hydrogen tartrate crystallization.

List of symbols mRG-II GC MS HPAEC HPSEC

Acknowledgments The authors thank Prs. A. Darvill and P. Albersheim, Dr. M. A. O’Neill, (University of Georgia, Athens, GA), Dr. R. Pe´pin (Rhoˆne-Poulenc Agro, Lyon, France), Dr. E. Samain (CERMAV-CNRS, Grenoble, France), and Dr. C. Plassard (INRA, Montpellier, France) for fruitfull discussion and advice. We thank M. J. Biron (INRA, Montpellier, France) for her assistance during the microbiological experiments, and J.-P. Lepoutre (INRA, Montpellier, France) for the GC-MS analysis.

References 1. 2. 3. 4. 5.

6. 7. 8.

9. 10.

Conclusions The isolated strain of P. daleae able to grow by degrading mRG-II represents a potential source for new pectinases. The study and use of these enzymes should be of potential interest for providing new data for the determination of the complete primary structure of the RG-II molecule. These

Monomeric Rhamnogalacturonan II Gas chromatography Mass spectrometry High performance anion-exchange chromatography High performance size-exclusion chromatography

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