ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 10, pp. 11641170. © Pleiades Publishing, Ltd., 2013. Published in Russian in Biokhimiya, 2013, Vol. 78, No. 10, pp. 14831490.
Structural Characterization of Enzymatically Synthesized Dextran and Oligosaccharides from Leuconostoc mesenteroides NRRL B1426 Dextransucrase D. Kothari and A. Goyal* Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, India; fax: 3612582249; Email:
[email protected] Received April 3, 2013 Abstract—Leuconostoc mesenteroides NRRL B1426 dextransucrase synthesized a high molecular mass dextran (>2 × 106 Da) with ~85.5% α(1→6) linear and ~14.5% α(1→3) branched linkages. This high molecular mass dextran containing branched α(1→3) linkages can be readily hydrolyzed for the production of enzymeresistant isomaltooligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The βlinked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic. DOI: 10.1134/S0006297913100118 Key words: dextransucrase, dextran, Leuconostoc mesenteroides NRRL B1426, acceptor, oligosaccharide
Dextransucrases (EC 2.4.1.5) elaborated by various strains of Leuconostoc mesenteroides, Streptococcus spp., Weisella spp., and Lactobacillus spp. synthesize dextrans or related glucans from sucrose [1]. Dextrans are homopolysaccharides of Dglucose units that are α (1→6) linked in the main chains with various degree of α (1→2), α(1→3), or α(1→4) branched linkages [2, 3]. The exact structure of the dextran depends on the specif ic dextransucrase produced by a bacterial strain, though the reaction conditions also contribute to the properties of the final product [4, 5]. Dextrans are used in the food industry as stabilizing, emulsifying, viscosifying, gelling, or waterbinding agents, and they also find a large appli cation in the area of nonfood industries [6]. A current consumer trend toward healthy and additivefree food has made dextrans an attractive solution for the replacement of commercial hydrocolloids used in food applications [7]. In addition to synthesizing glucans, dextransucrases also catalyze transglycosylation reactions, commonly referred to as acceptor reactions, in the presence of other carbohydrates. The acceptor reactions are secondary but * To whom correspondence should be addressed.
have been shown to compete with the synthesis of the dextran chains and even prevent their formation if a high enough concentration of the acceptor is present. This reaction is useful in (chemo)enzymatic synthesis of oligosaccharides as it prevents all kind of difficulties regarding stereo and regiospecificity associated with the chemical synthesis of (hybrid) carbohydrate compounds [810]. In general, functional oligosaccharides have a degree of polymerization (DP) from 2 to 8 monosaccha ride units [11]. Oligosaccharides have been increasingly used by the food industry (beverages, sweets) for modify ing viscosity, emulsification capacity, gel formation, freezing point, and color of foods. They also provide nutrition and healthrelevant benefits by modulating the composition of the colonic microbiota [12]. Moreover, the consumption of functional oligosaccharides reduces the risk of cardiovascular diseases, colon cancer, and obe sity [13]. The properties of oligosaccharides are highly dependent on the linkages of dextran produced by the dextransucrase and the type of acceptor molecule used. The efficiency and the extent of the acceptor reaction also vary with the particular acceptor molecule used [14]. Keeping the significance and the applications of dex tran and oligosaccharides in mind, the aim of the current
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DEXTRAN AND OLIGOSACCHARIDES FROM Leuconostoc mesenteroides NRRL B1426 DEXTRANSUCRASE 1165 study was to characterize dextran and oligosaccharides produced from L. mesenteroides NRRL B1426 dextran sucrase. The structure of the dextran was characterized by Fourier transform infrared (FTIR), 1H,13C nuclear mag netic resonance (NMR) spectroscopy, and gel perme ation chromatography (GPC). The oligosaccharides pro duced by the acceptor reaction of dextransucrase were analyzed using thin layer chromatography (TLC), high performance anion exchange chromatography (HPAEC), and electrospray ionization–time of flight mass spec trometry (ESITOF MS).
MATERIALS AND METHODS Microorganisms and chemicals. The strain L. mesen teroides NRRL B1426 was procured from the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, USA. The culture was maintained in modified de Man, Rogosa, and Sharpe (MRS) medium containing 20 g sucrose, 5 g yeast extract, 10 g peptone, 10 g beef extract, 2 g K2HPO4, 2 g triammonium citrate, 5 g CH3COONa, 1 ml Tween 80, 0.2 g MgSO4·7H2O, 0.2 g MnSO4·H2O, and 15 g agar per liter (pH 6.4) at 4°C and subcultured every 15 days [15]. LArabinose (LAra), D arabinose (DAra), Dcellobiose (Cel), Dfructose (Fru), Dgalactose (Gal), Dgentiobiose (Gnt), Dglucose (Glc), Disomaltose (Imal), Dlactose (Lac), Dmaltose (Mal), Dmannose (Man), Dmelibiose (Mel), Draffi nose (Raf), Drhamnose (Rha), Dtrehalose (Tre), and Dxylose (Xyl) were purchased from SigmaAldrich (USA). All other chemicals were of analytical grade. Production and estimation of dextran. Dextran was produced by incubating 1 ml PEG400 purified dextran sucrase (0.75 mg protein/ml, specific activity 10.1 U/mg) in 50 ml of 146 mM sucrose in 20 mM sodium acetate, pH 5.6, containing 0.3 mM CaCl2 and 15 mM NaN3 at 30°C for 24 h. The dextran produced was purified by ethanol precipitation [16], estimated by the phenol–sul furic acid method [17], and lyophilized for further analy sis. Monosaccharide analysis. Dextran (2 mg/ml) was hydrolyzed in 2 M trifluoroacetic acid (TFA) at 100°C for 4 h [18]. After removing the TFA, the released monosac charides were analyzed using a Dionex model ICS 3000 HPAEC system using a CarboPac P20 column (3 × 150 mm) with isocratic elution using 0.1 M NaOH at constant flow rate of 0.5 ml/min at 30°C. The monosac charide was detected with an electrochemical detector (ED 50). Glucose and fructose were used as standards. FTIR, NMR, and GPC analyses. The FTIR spec trum of the purified dextran was recorded in a KBr pellet using a PerkinElmer Spectrum One spectrometer for the identification of functional groups, monomeric units, and linkages. BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
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H and 13CNMR spectra for the dextran were recorded at 30°C with a 400 MHz Varian model AS400 NMR spectrometer equipped with VnmrX for Sun Microsystems v.6.1 software (operating frequencies 400 MHz for 1H and 100 MHz for 13CNMR) to deter mine the type of glycosidic linkage. The samples were dis solved in D2O (99.96%) at concentrations of 5 mg/ml (for 1 HNMR) and 30 mg/ml (for 13CNMR). The average molecular mass of the dextran was determined by GPC using an XK16/40 column (GE Healthcare) packed with Sephacryl S500HR on a GE Healthcare model 100 Plus AKTA purifier. The column was eluted using Milli Q water (18.2 MΩ) at constant flow rate of 1 ml/min. Dextran 500 and 2000 kDa were used as standards. Production of oligosaccharides. The oligosaccharides were produced by incubating 100 µl of PEG400 purified dextransucrase (0.75 mg protein/ml, specific activity 10.1 U/mg) [19] in 900 µl of the 16 different acceptors. The acceptortosucrose ratio was taken as 1 : 1 at 146 mM concentration in 20 mM CH3COONa (pH 5.6) containing 0.3 mM CaCl2 and 15 mM NaN3 at 30°C for 24 h. Absolute ethanol (2 ml) was added to the reaction mixture, and it was centrifuged at 16,000g for 10 min to remove polysaccharides (dextran). The precipitated dex tran was analyzed by the phenol–sulfuric acid method [17] and compared with the reaction with sucrose (5% w/v) without any acceptor. The supernatant contain ing oligosaccharides was analyzed using TLC, HPAEC, and ESITOF MS. The supernatant was diluted 200fold with Milli Q water and filtered through a 0.2 µm mem brane before analysis by HPAEC and ESITOF MS. Thin layer chromatography (TLC). An aliquot of 0.5 µl of each sample was absorbed onto a TLC plate (Merck 60; Germany), dried and developed in a solvent mixture composed of ethyl acetate–acetonitrile–H2O– 1propanol (2 : 7 : 5.5 : 5 v/v) [20]. After development, the carbohydrates were visualized by dipping the TLC plate into an ethanol solution containing 0.5% (w/v) α naphthol and 5% (v/v) H2SO4. After air drying, the TLC plate was placed into an oven at 120°C until the spots were visible [21]. High performance anionexchange chromatography (HPAEC). The oligosaccharides were analyzed quantita tively by an HPAEC system (model ICS 3000; Dionex) with ED 50 using a CarboPac P200 column (2 × 250 mm) by isocratic elution using 0.2 M NaOH at con stant flow rate of 0.5 ml/min at 30°C. Concentrations of oligosaccharides were calculated from peak areas using glucose as the standard [22]. Oligosaccharide production was compared with the maltose acceptor reaction (100%). Mass spectrometry. The oligosaccharides were iden tified with an ESITOF MS (Waters model QTOF Micromass HAB273) using the following ion source parameter conditions: sourcedesolvation gas tempera
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tures set at 100 and 250°C, respectively; sampling, capil lary, and extraction cone voltages were set 35 V, 3 kV, and 3 V, respectively, with ion guide of 1 V. Flow injection rate was set at 10 µl/min. Mass spectra were acquired in posi tive ion mode and processed using MassLynx software.
RESULTS AND DISCUSSION Monosaccharide analysis. Monosaccharide analysis of L. mesenteroides NRRL B1426 dextran showed that it was composed of only Dglucose residues, indicating its glucan nature (data not shown). Spectroscopic analyses. The type of linkages and the functional groups of the dextran from L. mesenteroides NRRL B1426 were characterized by FTIR spectroscop ic analysis (Fig. 1). The fingerprint region for polysaccha ride is considered to be from 1200950 cm–1 for their pos sible identification based on the position and intensity of the bands [23]. The dextran showed a peak at 1018 cm–1, which is characteristic for α(1→6) linkages as reported by Shingel [24]. The αglycosidic bond was confirmed by the presence of a peak at 916 cm–1. The band at 1153 cm–1 was caused by covalent vibrations of the C–O–C bond and glycosidic bridge. There was no characteristic peak at 890 cm–1, this indicating the absence of βconfiguration.
The hydroxyl and the C–H stretching vibrations corre sponded with the bands in the region of 3440 and 2922 cm–1, respectively. The band in the region of 1651 cm–1 was due to bound water. Similar results were reported by Purama et al. [16] for dextran from L. mesen teroides NRRL B640. Thus, the FTIR spectrum revealed that the dextran from L. mesenteroides NRRL B1426 contains α(1→6) linkages, which was further confirmed through 1H and 13C NMR analyses. The 1H and 13C NMR spectra display the main struc tural features of the dextran. Sidebotham [25] reported that various dextrans have 1H NMR spectral resonances (C2, C3, C4, C5, and C6) in the 34 ppm region and the hemiacetal C1 resonance in the 46 ppm region. The dextran showed 1H chemical shifts of anomeric signals at 4.96 ppm and a low intensity peak at 5.33 ppm (Fig. 2a), which were due to α(1→6) and α(1→3) glycosidic link ages, respectively. Similar results were obtained by Seymour et al. [26] for dextran from L. mesenteroides NRRL B1355 and van Leeuwen et al. [27] for Lactobacillus reuteri. Other protons appeared as a com plex series of overlapping signals ranging from 3.4 to 4 ppm. The peak recorded at 4.8 ppm in the spectrum was from D2O (Fig. 2). Based on integration analysis of the 1H NMR signal, it was inferred that the dextran from L. mesenteroides NRRL B1426 contains ~85.5% α(1→6)
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DEXTRAN AND OLIGOSACCHARIDES FROM Leuconostoc mesenteroides NRRL B1426 DEXTRANSUCRASE 1167
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Cel Glc Gnt Imal Lac Mal Mel Tre Suc Carbohydrates
Fig. 4. Enzymatic synthesis of oligosaccharides (black columns) and dextran (gray columns) from L. mesenteroides NRRL B1426; Cel, cellobiose; Glc, glucose; Gnt, gentiobiose; Imal, isomaltose; Lac, lactose; Mal, maltose; Mel, melibiose; Tre, trehalose; Suc, sucrose. The data are expressed as the mean of triplicates ± stan dard deviation.
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linked Dglucosyl linkages and ~14.5% α(1→3) linked Dglucosyl linkages. There were 145 branched linkages for every 1000 glucose units of glucan. The presence of no other signal in the region 4.95.3 ppm, except at 5.33 ppm in the 1H NMR spectrum, indicated the absence of any
branching other than α(1→3) in the dextran from L. mesenteroides NRRL B1426. Dextrans have their 13C NMR anomeric signals downfield at ~90 ppm, while C2, C3, C4, and C5 appear in the 7085 ppm range, and C6 is normally
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m/z Fig. 5. ESITOF MS of effective acceptor reaction mixtures: a) cellobiose; b) gentiobiose; c) glucose; d) isomaltose; e) lactose; f) maltose; g) melibiose; h) trehalose.
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DEXTRAN AND OLIGOSACCHARIDES FROM Leuconostoc mesenteroides NRRL B1426 DEXTRANSUCRASE 1169 upfield at ~60 ppm [28]. The 13C NMR spectrum of the L. mesenteroides NRRL B1426 dextran showed six prominent resonances as shown in Fig. 2b, which were characteristics of linear dextrans as reported by Seymour et al. [29]. Apart from these six peaks, which correlate with six signals of linear dextran, the spectrum also con tained minor peaks indicative of branching. The signal at 60.5 ppm assigned to the C6 atom on the nonreducing glucose units was of considerable interest as it corre sponded to branching [30]. The resonance peaks at 99.5 and 80.8 ppm indicated branched linkage at C3, which was also supported by resonance at 5.33 ppm in the 1H NMR spectrum. Similar results of branching of α(1→3) linkage were also reported for dextran from L. mesen teroides NRRL B512F at 99.5 ppm by Colson et al. [30] and for dextran from L. citreum NRRL B742 at 81.66 ppm by Seymour and Knapp [31]. Several studies have previously reported that the content of α(1→6) glyco sidic linkages in dextrans originating from lactic acid bac teria ranges from 50 to 100%. The diversity observed in the structures and properties of bacterial dextrans could be attributed to differences between microbial strains and variations in growth rates and reaction conditions [32]. Thus, the 1H and 13C NMR spectral analyses confirmed that the L. mesenteroides NRRL B1426 dextran was composed of ~85.5% α(1→6) linear and ~14.5% α (1→3) branched linkages. GPC analysis. The average molecular mass of the dextran from L. mesenteroides NRRL B1426 was found to be >2 × 106 Da by GPC (Fig. 3). High molecular mass dextrans of (12) × 106 Da have been approved by the European Union as food ingredients in bakery products [33]. The required molecular mass has been reported to be from 2 × 106 to about 4 × 106 Da [34]. Relatively high molecular mass dextrans have been crosslinked to vary ing extents to give different sizeexclusion chromatogra phy materials (Sephadex products) that have been used as gel filtration materials for over 50 years [1]. Production and analysis of oligosaccharides. The acceptor reaction of dextransucrase from L. mesenteroides NRRL B1426 with 16 different sugars was investigated. TLC analysis showed that cellobiose, gentiobiose, glu cose, isomaltose, lactose, maltose, melibiose, and tre halose are effective acceptors for oligosaccharide synthe sis (data not shown). However, no acceptor reaction products were obtained with arabinose, fructose, galac tose, mannose, rhamnose, raffinose, and xylose. The effective acceptors were further compared for their effi ciency towards acceptor reaction by monitoring the oligosaccharide concentrations. The best acceptor for oligosaccharide production after maltose (100%) was iso maltose (89%), followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%). Dextran production was observed, and it could not be avoided in the reaction of dextransucrase with any acceptor (Fig. 4). ESITOF MS BIOCHEMISTRY (Moscow) Vol. 78 No. 10 2013
analysis was also performed to confirm the effective acceptors for oligosaccharide synthesis. ESITOF MS analysis of oligosaccharides with vari ous acceptors revealed predominant ion species as sodi um or potassium adducts [M+Na]+ or [M+K]+ as shown in Fig. 5. The sodium or potassium adducts of oligosac charides ranging from DP3 to DP6 (5271013 or 543 1029) were observed for all the acceptors. However, the peak intensities of sodium or potassium molecular ions of DP > 4 (6891013 or 7051029) varied with the type of acceptor molecule used. Oligosaccharides of DP > 4 were present in considerable amount in the case of gentiobiose, isomaltose, lactose, and maltose (Fig. 5, b, d, e, and f). However, DP3 and DP4 were the main acceptor products with cellobiose, glucose, melibiose, and trehalose (Fig. 5, a, c, g, and h). Thus, gentiobiose, lactose, and isomaltose could also be used as efficient acceptors other than malt ose for oligosaccharide production using dextransucrase from L. mesenteroides NRRL B1426. Kim et al. [35] described that the product composition of acceptor reac tions could also vary with donor/acceptor ratio. Since β linkages are stronger than αlinkages and are highly resistant to the attack of digestive enzymes [36], cel lobiose (β1→4 linked), gentiobiose (β1→6 linked), and lactose (β1→4 linked) could act as efficient acceptors for prebiotic oligosaccharide synthesis. In summary, high molecular mass dextran from L. mesenteroides NRRL B1426 dextransucrase is a candi date for use in food industries. The prebiotic properties of oligosaccharides are governed by their linkages and, hence, the acceptor products from L. mesenteroides NRRL B1426 dextransucrase can be tailored to meet specific industrial requirements in nutritional research. This work was supported by a project grant from the Department of Biotechnology, Ministry of Science and Technology, New Delhi, India to AG. The authors grate fully acknowledge the Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, USA for providing the microorganism and also the Central Instrument Facility, Indian Institute of Technology Guwahati, Guwahati, for NMR and ESITOF MS facilities.
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