Purification and structural characterisation of (1→3;1→6)-β--glucans (botryosphaerans) from grown on sucrose and fructose as carbon sources: a comparative study

Purification and structural characterisation of (1→3;1→6)-β--glucans (botryosphaerans) from grown on sucrose and fructose as carbon sources: a comparative study
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  Purification and structural characterisation of (1 / 3;1 / 6)- b - D -glucans(botryosphaerans) from  Botryosphaeria rhodina  grown on sucrose andfructose as carbon sources: a comparative study Maria de Lourdes Corradi da Silva a, *, Nataly L. Izeli a , Paula F. Martinez a , Iara R. Silva a ,Carlos J.L. Constantino a , Marilsa S. Cardoso a , Aneli M. Barbosa b , Robert F.H. Dekker b ,Gil V.J. da Silva c a  Departamento de Fı´ sica, Faculdade de Cieˆ ncias e Tecnologia, Quı´ mica e Biologia, Universidade Estadual Paulista, CEP 19060-900,Presidente Prudente, SP, Brazil b  Departamento de Bioquı´ mica e Biotecnologia, Universidade Estadual de Londrina, CCE, Caixa Postal 6001, CEP 86051-990, Londrina, PR, Brazil c  Departamento de Quı´ mica, Faculdade de Filosofia, Cieˆ ncias e Letras, Universidade de Sa˜ o Paulo, CEP 14040-901, Ribeira˜ o Preto, SP, Brazil Received 18 November 2004; revised 9 January 2005; accepted 12 January 2005Available online 13 May 2005 Abstract Two botryosphaerans, exopolysaccharides (EPS) secreted by the ascomyceteous fungus  Botryosphaeria rhodina , when grown on sucroseand fructose as sole carbon sources, were structurally compared after their isolation from the culture medium. Both EPS were submitted totrypsin digestion, and eluted as a single peak on gel filtration. Total acid hydrolysis yielded only glucose, and data from methylation analysisand Smith degradation indicated that both EPS constituted a main chain of glucopyranosyl b (1 / 3) linkages substituted at O-6. The productsobtained after partial acid hydrolysis demonstrated side chains consisting of glucosyl- and gentiobiosyl- linked  b (1 / 6) residues.  13 C-NMRspectroscopy studies showed that all glucosidic linkages were of the  b -configuration. The carbon source affected the side chain structures of botryosphaeran but not the main chain makeup. Sucrose produced less branching (21%) than fructose (31%). q 2005 Published by Elsevier Ltd. Keywords: Botryosphaeria rhodina ; Exopolysaccharides;  b (1 / 3;1 / 6)- D -Glucans; Carbon source; Structural variation 1. Introduction Industrial applications of polysaccharides have reliedmainly upon raw materials from plants and marine algaeuntil recently (Tharanathan, 1995). Nowadays, fungalpolysaccharides are receiving increased attention due totheir peculiar physicochemical and rheological properties,concomitant with novel functionality (Lowman, Ferguson,& Williams, 2003; Ricciardi et al., 2002; Sandula, Kogan,Kacura´kova´, & Machova´, 1999; Selbmann, Onofri, Fenice,Federici, & Petruccioli, 2002). Consequently, microbialpolysaccharides as biomaterials have found a wide range of new applications including use in pharmaceutical therapydue to their unique physiological activities; as antitumor,antiviral and anti-inflammatory agents (Ishibashi et al.,2004; Kumar, Joo, Choi, Koo, & Chang, 2004; Li et al.,2004; Tsiapali et al., 2001). Many of the biological activitiesof exopolysaccharides (EPS) such as the  b -glucans areattributable to the (1 / 3) type of linkage (Alquini,Carbonero, Rosado, Consentino, & Iacomini, 2004; Bao,Zhen, Ruan, & Fang, 2002; Engstad, Engstad, Olsen, &Osterud, 2002; Falch, Espevik, Ryan, & Stokke, 2000;Kulicke, Lettau, & Thielking, 1997; Willment, Gordon,& Brown, 2001). They are usually structurallycomplex homopolymers comprised of glucose that aregenerally isolated from yeasts and fungi. Differentphysicochemical parameters of   b -glucans, such as solubi-lity, primary structure, molecular weight, extent of  Carbohydrate Polymers 61 (2005) 10–$ - see front matter q 2005 Published by Elsevier Ltd.doi:10.1016/j.carbpol.2005.01.002* Corresponding author. Tel.: C 55 18 229 5355x26; fax: C 55 18 2215682. E-mail address: (M. de Lourdes Corradi daSilva).  branching by side-chain substituents (Bohn & BeMiller,1995), and the charge on the polymer, all appear to influencetheir biological activity (Vetvicka & Yvin, 2004). Schizo- phyllan, one of the  b (1 / 3)- D -glucans known to haveimmunomodulating potential and antitumor activity, hasbeen used clinically as an immunopotentiator against sometypes of cancer, chiefly leukocytopenia (Borchers, Stern,Hackman, Keen, & Gershwin, 1999; Kidd, 2000; Kubala etal., 2003).Although many fungal  b (1 / 3)-glucans have beendescribed, only a few have been rigorously characterised.Consequently, their chemical diversity and potentiallyuseful functional properties are still poorly understood(Schmid et al., 2001). Since these EPS occur in Nature asmixtures of heterogeneous cellular components orsecretions, it is first necessary to isolate, purify andstructurally characterise these polysaccharides, beforeproceeding with detailed investigations of their propertiesand applications (Kim et al., 2003; Sutherland, 1998). The ascomyceteous fungus  Botryosphaeria  sp. (isolateMAMB-05)hasbeenstudiedasalaccaseproducersince1995(Barbosa, Dekker, & Hardy, 1996; Barbosa, Dekker,Kurtbo¨ke, & Hardy, 1995; Dekker, Vasconcelos, Barbosa,Giese, & Paccola-Meirelles, 2001), and was only recentlyclassified to the species level as:  Botryosphaeria rhodina (Garcia, Vilas-Boas, Dekker, Fungaro, & Barbosa, 2004)using molecular biology techniques. However, when thisligninolytic fungus was grown on glucose as sole carbonsource,anEPSofthe b -glucantypewassecretedintheculturemedium (Dekker & Barbosa, 2001). The structure of this glucanwascharacterised(Barbosa,Steluti,Dekker,Cardoso,& Corradi da Silva, 2003) recently as a (1 / 3;1 / 6)- b - D -glucan with approximately 22% side branching at C-6. Thebranches consisted of single (1 / 6)- b -linked glucosyl, and(1 / 6)- b -linked di-glucosyl (gentiobiose) residues. This  b -glucan type was subsequently named botryosphaeran inaccordance with the fungus producing this EPS type.A comparison of botryosphaeran production by  B. rhodina  on several carbohydrate carbon sources hasrecently been described (Steluti et al., 2004). With theexception of mannitol, the fungus produced EPS on all of the carbon sources examined, with highest yields occurringwith sucrose followed by glucose and fructose. AccordingtoJin et al. (2003) and Zhang, Yang, Ding, and Chen (1995),different carbon sources generate similar bioactivepolymers with different degrees of branching and distinctpolymerisation, producing biopolymers that are more or lesswater-soluble, and as a consequence, may possess higher orlower biological activity.The work reported herein discusses the purification andstructural characterisation of the botryosphaerans producedby  B. rhodina  when the fungus was grown on fructose(EPS FRU ) and sucrose (EPS SUC ).  13 C-NMR spectroscopystudies showed that all glucosidic linkages were of the b -configuration and that the carbon source affected onlythe side chain structures of the botryosphaerans but not themain chain constitution. 2. Materials and methods 2.1. Microorganism, culture media and growth conditions Botryosphaeria rhodina  was maintained at 4  8 C onpotato-dextrose-agar (Barbosa et al., 1996). Inoculum wasprepared by growing  B. rhodina  on minimum saltsmedium (Vogel, 1956) containing agar (20 g/L) and glucose(10 g/L). After 5 days growth (28  8 C), mycelial fragmentswere transferred to four 125 mL Erlenmeyer flasks contain-ing 25 mL of minimum salts medium and glucose (0.5 g/L),and incubated at 28  8 C for 48 h on a rotary shaker(180 rpm). The mycelia were homogenised for 30 s atmaximum speed. Then, the cell homogenate was centri-fuged for 10 min at 1250 ! g , the mycelium recovered, anddiluted with sterilised physiological saline solution to anabsorbance between 0.4 and 0.5 at 400 nm. Four millilitre-aliquots of the suspension were used to inoculate Erlen-meyer flasks (1 L) containing 200 mL of minimumsalts medium and sole carbon sources (sucrose and fructose,5 g/L). Cultures were grown in submerged cultivation(180 rpm) for 72 h at 28  8 C. 2.2. Preparation and dissolution of exopolysaccharides Cell-free extracellular fluid was obtained after removalof the mycelium by centrifugation (5500 ! g  /10 min) at4  8 C. The supernatant was treated with 3 volumes of absolute ethanol, the precipitate recovered and dissolved indistilled water, followed by extensive dialysis againstfrequent changes of distilled water, and then freeze-dried.This preparation was used to determine the carbohydrateand protein content. 2.3. Analytical techniques Total sugars were determined by the phenol–sulphuricacid method of  Dubois, Gilles, Hamilton, Rebers, andSmith (1956) and reducing sugars were quantified by thecupro-arsenate method of Somogyi–Nelson (Nelson, 1944).Glucose was used as the standard in both assayprocedures. Protein was measured by the Bradford method(1976) using bovine serum albumin as standard, andspectrophotometrically at 280 nm. 2.4. Purification of botryosphaerans A sample of each botryosphaeran (100 mg; EPS SUC  andEPS FRU ) was dissolved in 0.1 M sodium phosphate buffer(150 mL, pH 7.6) followed by the addition of 5 mg of trypsin (Sigma) enzyme (Krcmar, Novotny, Maraias, &Joseleau, 1999) and the solutions incubated at 37  8 C for  M. de Lourdes Corradi da Silva et al. / Carbohydrate Polymers 61 (2005) 10–17   11  48 h. After the digestion step, the samples were extensivelydialysed against frequent changes of distilled water andfreeze-dried. The procedure was repeated twice, andprotein, total and reducing sugars were determined onthese samples to assess homogeneity. The EPS preparationswere then dissolved in water (1 g/L) and purified by gelpermeation chromatography on a column of SepharoseCL-4B (43.0 cm ! 1.0 cm) and eluted with water (0.3 mL/ min). Fractions (2.6 mL) were collected and analysed forcarbohydrate and monitored at 280 nm for protein. Frac-tions corresponding to peaks were pooled and freeze-dried. 2.5. Monosaccharide analysis Polysaccharide samples (0.050 mg) were hydrolysed in0.5 mL of 5 M trifluoroacetic acid (TFA) at 100  8 C for 16 h(Barbosa et al., 2003). After hydrolysis, the solutions wereevaporated under vacuum, and the residue dissolved in0.5 mL of water and evaporated again. The dissolution–evaporation cycle was repeated twice. Finally, theresidue was dissolved in 0.2 mL of water, and 0.025 mLaliquots used for High Performance Anionic ExchangeChromatography with Pulsed Amperometric Detection(HPAEC/PAD); Dionex Chromatograph DX 500 (Fan,Namiki, Matsuoka, & Lee, 1994; Weitzhandler et al.,1993). Neutral monosaccharides were separated isocrati-cally (0.014 M NaOH) using a CarboPac PA-10 (DionexChromatography) column (4 ! 250 mm) equipped with aPA-10 guard column at a flow rate of 1.0 mL/min (Elı´fio,Corradi da Silva, Iacomini, & Gorin, 2000; Weitzhandleret al., 1993). Elution was performed using water (eluent 1)and 0.2 M NaOH (eluent 2). The column was regeneratedafter 20 min using 100% of eluent 2 for 15 min, followed bya return to 0.014 M NaOH. Monosaccharide quantificationwas carried out from peak area measurements usingresponse factors obtained from standard monosaccharides. 2.6. Methylation–acid hydrolysis–acetylation Fractions of each botryosphaeran (10 mg) were solubil-ised in dimethyl sulfoxide (1 mL), methylsulfinylcarbanion(0.5 mL) was added, and the mixture sonicated for 50 min atambient temperature followed by the gradual addition of methyl iodide (0.3 mL). The methylated polysaccharideswere extracted with chloroform (4 mL), and the chloroformphase evaporated until completely dry (Barbosa et al.,2003). Second and third methylationruns were performed asabove. The permethylated polysaccharides were solubilisedin 72% (v/v) H 2 SO 4  (0.5 mL) in an ice bath for 2 h, and thenwater was added (4 mL) to obtain a final concentration of 1.4 M and kept under reflux at 100  8 C for 18 h (Bouveng &Lindberg, 1965; Corradi da Silva, Iacomini, Jablonski, &Gorin, 1993). The resulting partially methylated sugarswere reduced with NaBH 4 , acetylated, and analysed bygas liquid chromatography (GLC) on a Varian Model 3300Gas Chromatograph. A conventional column DB-225(medium-polar column—silicon polymer containingmethyl, phenyl and nitrite groups) was used. The injectiontemperature was 50  8 C with a program to 220  8 C (constanttemperature) (Woranovicz, Pinto, Gorin, & Iacomini,1999). 2.7. Smith degradation Samples (40 mg) of each botryosphaeran were dissolvedin 1 M NaOH (7 mL) and 0.5 M HCl added to a final pH of 4.6. Each sample was then oxidised with aqueous 0.05 MNaIO 4  (50 mL) for 96 h at 4  8 C in the dark (Fabre,Bruneteau, Ricci, & Michel, 1984). The oxidised poly-saccharides were next reduced with 1.0 M NaBH 4 , and aportion subjected to total acid hydrolysis (1.0 M TFA,100  8 C, 6 h). Another portion was subjected to mild acidhydrolysis (1.0 M H 2 SO 4 , 24 h, 50  8 C) to remove theoxidised sugar residues attached to the polysaccharidechain (Smith degradation), followed by neutralisation,dialysis against water and freeze-drying. A sample of eachoxidised botryosphaeran product (5 mg) was methylatedtwice as described above, and another portion (6.0 mg) wassubjected to  13 C-NMR spectroscopy. 2.8.  13 C-NMR spectroscopy 13 C-NMR spectra were obtained on a Bruker DRX-400NMR Spectrometer with each botryosphaeran (10–12 mg)sample in dimethyl sulfoxide at 400 MHz (30  8 C). Chemicalshifts were referred to tetramethylsilane (Gorin, 1981). The high viscosity of the EPS presented problems affecting thequality of the NMR spectra. 2.9. Partial acid hydrolysis Partial acid hydrolysis of each botryosphaeran (30 mg)was performed as described by Ukai, Yokoyama, Hara, andKiho(1982)using50%(v/v)H 2 SO 4 for16 hat4  8 C,andthenstirred for 1 h at 35  8 C. The hydrolysed material wasneutralised with barium carbonate, de-ionised with Amber-liteIR-400(carbonate)resin,andtheproductsseparatedonacolumnofSephadexG-15(103 cm ! 0.8 cm)calibratedwithstarch, melibiose and glucose. Water was used as eluent(0.18 mL/min) and fractions collected were analysed forcarbohydrate by the phenol–sulphuric acid method. Frac-tions containing the modified poly-, oligo- and mono-saccharides were analysed by HPAEC/PAD (Lee & Rice,1993; Rice & Corradi da Silva, 1996). Mono- and oligo- saccharides were separated on a CarboPac PA-100 column(4 ! 250 mm)andguardcolumn(4 ! 50 mm)ataflowrateof 1 mL/min. The column was equilibrated in 0.1 M NaOH(97%) and 0.5 M sodium acetate (3%). After 15 min, a0–0.25 Msodiumacetategradientwasappliedovera60 mininterval, while the concentration of NaOH remained at0.1 M.Monosaccharidesandoligosaccharidesweredetectedby Pulsed Amperometric Detection (Dionex DX-500)  M. de Lourdes Corradi da Silva et al. / Carbohydrate Polymers 61 (2005) 10–17  12  withouttheadditionofpost-columnalkali.Monosaccharidesand oligosaccharides in the experimental samples wereidentified by comparison to known retention times (min).Theretentiontimes( T  R )ofthestandardsugarswere:glucose(2.86 min), gentiobiose (4.58 min), laminaribiose(7.50 min), gentiotriose (11.55 min) and laminaritriose(15.01 min). 3. Results and discussion Quantification of total sugars (an indication of EPScontent), reducing sugars and protein in the botryosphaeransamples, EPS FRU  and EPS SUC , revealed a high content of carbohydrateinrelationtoprotein.Thecarbohydrate/proteinratios for EPS FRU  and EPS SUC  were 1:0.035 and 1:0.123,respectively. It is not uncommon for EPS preparations tocontain protein, which co-precipitates with polysaccharidesduring extraction with ethanol, and can be removed bydigestion with proteases. This was the case for a crude EPSpreparation from  Phlebia radiata  containing 20% proteinand 80% carbohydrate, and was purified following trypsindigestion resulting in a protein-free material characterised(Krcmar et al., 1999) as a (1 / 3;1 / 6)- b - D -glucan. Bothbotryosphaerans from  B. rhodina  when subjected to trypsindigestion resulted in highly enriched carbohydrate-contain-ing material following this treatment, and gave a carbo-hydrate/protein ratio of 1:0.003 and 1:0.001, respectively.Aliquots of the trypsin-digested botryosphaeran samplesfractionated by gel permeation chromatography on aSepharose CL-4B column resulted in each EPS eluting as asingle peak (Fig. 1) indicating homogeneity. These prep-arations were then considered adequately pure for structuralcharacterisation. After total acid hydrolysis of both purifiedbotryosphaeran fractions, only glucose was detected byHPAEC analysis. This finding was similar to the EPSproduced when  B. rhodina  was grown on glucose as solecarbon source (Barbosa et al., 2003).When submitted to methylation-GLC analysis to deter-minethenatureoftheglucosidiclinkage,theEPSshowedthefollowing methylated sugar derivatives: (I) 2,3,4,6-tetra- O -methyl-glucose corresponding to non-reducing terminalunits, (II) 2,4,6-tri- O -methyl-glucose correlated to 3-O-substituted glucosyl residues, (III) 2,3,4-tri- O -methyl-glu-cose correlated to 6-O-substituted glucosyl residues, and(IV) 2,4-di- O -methyl-glucose attributed to 3,6-di-O-substi-tuted units, in different molar proportions for each botryo-sphaeran sample (Table 1). These results established thatboth botryosphaerans constituted a (1 / 3)-linked glucosylbackbone substituted with branch points on C-6. Thereduced content of component III suggests that thebackbone essentially consisted of consecutive glucoseresidues (1 / 3)-linked. The relatively low amount of component II in EPS FRU  in comparison to derivatives I andIV, is in agreement with a highly branched structure, whilethat for EPS SUC  was somewhat lower. Structural studies onthe extracellular  b - D -glucans from  Phytophthora parasitica (Gandon & Bruneteau, 1998) by the Hakomori methodshowed similar methylated sugar derivatives in differentmolar ratios, establishing that these glucans consisted of (1 / 3)-, (1 / 6)-, and (1 / 3;1 / 6)- linkages, and non-reducingterminalglucopyranosidecomponentshavingalowproportion of (1 / 6)-linked residues. The relatively lowamountofthe2,4,6-tri- O -methylderivativeincomparisontothe tetra- O -methyl and the di- O -methyl derivatives agreedwith a highly branched structure, and the low value of  Fig. 1. Gel permeation chromatography profile of botryosphaerans EPS FRU and EPS SUC  from  Botryosphaeria rhodina  on a Sepharose CL 4B columnafter trypsin digestion. The column (43.0 cm ! 1.0 cm) was eluted withwater at a flow rate of 0.3 mL/min. ( † ) A 625 : Blue Dextran; ( ! ) A 490 :EPS FRU ; ( : ) A 490 : EPS SUC .Table 1Methylation analysis of the botryosphaerans produced by  Botryosphaeriarhodina  cultured on sucrose and fructose as carbon sourcesMethylated derivatives Molar ratios (%)EPS FRUa EPS SUCa 1,5-Di- O -acetyl-2,3,4,6-tetra- O -methyl glucitol 34 231,3,5-Tri- O -acetyl-2,4,6-tri- O -methyl glucitol 20 471,5,6-Tri- O -acetyl-2,3,4-tri- O -methyl glucitol 15 91,3,5,6-Tetra- O -acetyl-2,4-di- O -methyl glucitol 31 21 a Refers to the exopolysaccharide, Botryosphaeran, arising from thefungus grown on either fructose or sucrose.  M. de Lourdes Corradi da Silva et al. / Carbohydrate Polymers 61 (2005) 10–17   13  the 2,3,4-tri- O -methyl derivative suggested that the back-bonechainofthisglucanconsistedessentiallyofconsecutive(1 / 3)-linked  D -glucose residues. Methylation analysis of the exopolysaccharide from spores of   Ganoderma lucidum (Bao et al., 2002) yielded three derivatives: 2,3,4-tetra-, 2,4,6-tri-, and 2,4-di- O -methyl glucose (1:4:1), suggestingthat this glucan too contained a backbone chain composedessentially of (1 / 3) linkages with side branching on C-6.The botryosphaerans were subjected to sequentialperiodate oxidation and borohydride reduction, followedby total and partial acid hydrolysis. Total hydrolysis of theEPS SUC  released glycerol (35%), which corresponded tonon-reducing terminal and 6-O-substituted units, andglucose (65%) arising from 3-O- and 3,6-di-O-substitutedcomponents that were not oxidised by periodate. The samecomponents were also obtained from EPS FRU , but indifferent amounts, 54 and 46%, respectively. Mild hydroly-sis followed by GLC analysis indicated the almosttotal disappearance of non-reducing terminal units. Theevidence of (1 / 3)-linked glucans was confirmed bythe detection of the major component, 1,3,5-tri- O -acetyl-2,4,6-tri- O -methyl- D -glucitol. These structural character-istics have been found mainly in fungal polysaccharides(Krcmar et al., 1999; Woranovicz et al., 1999). The extracellular  b - D -glucans derived from  Phytophthora parasitica  (Gandon & Bruneteau, 1998) when subjected toSmith degradation followed by mild acid hydrolysis showed2,3,4,6-tetra-, 2,4,6-tri, and 2,4-di- O -methyl glucose (molarratios 1:7:1) by GLC analysis of their alditol acetatederivatives, which was in accordance with a (1 / 3)-linkedbackbone chain structure.In accordance with some literature reports,  13 C-NMRspectra of the botryosphaerans (Fig. 2, Table 2) showed signals attributable to  b (1 / 3)-glucans O-substituted onC-6 (Gorin, Baron, Corradi da Silva, Teixeira, & Iacomini,1993), but did not reveal peaks at  d  100.0 ppm thatcorresponded to the  a -configuration of the anomeric carbon(Schmid et al., 2001). Peaks were only visible between  d 103.3 and 102.9 ppm, which strongly indicated that only b -anomeric carbons were present. The signal at  d  103.1 wasattributed to the C-1 of 3-O-substituted glucopyranosyl unitsin comparison to a high rate of 2,4,6 tri- O -methylglucitolderivative obtained after Smith degradation. The signal at  d 102.9 was attributable to 3-O-substituted glucose units of the main chain containing branched residues ( b -glucopyr-anosyl and  b -di-glucopyranosyl) on C-6, in agreement withthe general rule for  b -anomeric carbon glycosylation thatexplained an upfield chemical shift. The signal at  d  103.3corresponded to the anomeric carbon of non-reducingterminal glucose units. Fig. 2.  13 C-NMR spectral assignments of botryosphaerans EPS FRU  and EPS SUC  produced by  Botryosphaeria rhodina .  M. de Lourdes Corradi da Silva et al. / Carbohydrate Polymers 61 (2005) 10–17  14
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