Structural elucidation of the exopolysaccharide produced by fungus Fusarium oxysporum Y24-2

Carbohydrate Research 365 (2013) 9–13

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Structural elucidation of the exopolysaccharide produced by fungus Fusarium oxysporum Y24-2 Shoudong Guo a,b,⇑, Wenjun Mao b, Yanling Li a, Jinghui Tian c, Jian Xu b a

Life Science Research Center, Taishan Medical University, Taian, Shandong 271000, People’s Republic of China Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology, Ocean University of China, Qingdao 266003, People’s Republic of China c School of public health, Taishan Medical University, Taian, Shandong 271000, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 30 July 2012 Received in revised form 26 September 2012 Accepted 28 September 2012 Available online 6 October 2012 Keywords: Structural elucidation Extracellular polysaccharide NMR spectroscopy

a b s t r a c t The extracellular polysaccharide FO1 was isolated from the fermentation broth of an endophytic fungus (Fusarium oxysporum) of Ipomoea pes-caprae. Its structural characteristics were studied by chemical and methylation analyses, and 1D and 2D 1H and 13C NMR spectroscopy. Results indicated that this exopolysaccharide consists of a disaccharide repeating unit with the following structure (n  111): [?2)-b-D-Galf(1?6)-a-D-Glcp(1?]n Ó 2012 Elsevier Ltd. All rights reserved.

1. Instruction Polysaccharides play important roles in pathogenic infection1,2 and are demonstrated to be useful chemotaxonomic markers.3 In general, elucidation of the chemical composition of these pathogenic fungi is the primary event for the preventive and epidemiological research. Some of the cell wall polysaccharides from Fusarium species have been reported, for instance, the chemical characteristics of the polysaccharide produced by Fusarium sp. M7-1,4 and the detailed cell wall structure of the acid polysaccharide.5–8 Recently, a group investigated the glucan differences of Fusarium strains by FT-IR spectroscopy.2 Previously, we reported some of the secondary metabolites produced by the endophytic fungus (Fusarium oxysporum Y24-2) of Ipomoea pes-caprae, and found they have good antitumor activity.9 The cell wall polysaccharides of Fusarium oxysporum have been reported.10,11 We herein report on the structure of a novel extracellular polysaccharide from the fermentation broth of Fusarium oxysporum Y24-2. 2. Experimental 2.1. Microbial strain and culture conditions Fusarium oxysporum Y 24-2 was isolated from Ipomoea pescaprae (Linn.).9 The basal medium and growth conditions were

⇑ Corresponding author. Tel.: +86 538 622 2706; fax: +86 538 622 5275. E-mail address: [email protected] (S. Guo). 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2012.09.026

similar to those described previously.12 Briefly, the fungus was cultivated in the liquid medium containing yeast extract (3 g/L), malt extract (3 g/L), peptone (5 g/L), glucose (20 g/L), KH2PO4 (0.5 g/L), NH4Cl (0.5 g/L), and pH 6.0–6.5 at 25 °C for 7 days on a reciprocal shaker.13 2.2. Isolation and purification The purification process was similar to the method we previously reported.14,15 FO1 containing eluate was obtained from an anion exchange column, Q-Sepharose Fast Flow (2.6  50 cm), taking distilled water as eluent. The distilled water fraction was concentrated and further purified on a Sephadex G150 column (2.0  90 cm) eluting with 0.2 mol/L ammonium bicarbonate at a flow rate of 0.2 mL/min. The corresponding fraction was pooled according to the result of phenol-H2SO4 method.16 2.3. Component analysis Sugar content was detected by the phenol-H2SO4 method with mannose as the standard.16 Protein content was measured by the method of Bradford using bovine serum albumin as the standard. Sulfate ester content was detected according to the method reported by Therho and Hartiala.17 Uronic acid content was detected by the carbazole–H2SO4 method using glucuronic acid as the standard.18 2.4. Monosaccharide composition analysis The neutral monosaccharide composition was analyzed according to the method of Chen et al.12 Gas chromatography was

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performed on a HP6890 instrument with a SE-54 fused silica capillary column (320 lm i.d. 50 m, Agilent Technology, USA) and flame-ionization detector. The operation was performed using the following conditions: H2: 1.5 ml/min; air: 200 ml/min; N2: 1.5 ml/min; detector temperature: 250 °C; column temperature: 212 °C. Sugar identification was done by comparison with reference sugars. 2.5. Measurement of homogeneity and molecular weight Molecular weight was measured by high performance gel permeation chromatography (Shodex OHpak SB-804 HQ column) and a refractive index detector. The column and the detector were maintained at 35 °C, and the mobile phase was 0.1 mol/L sodium sulfate at a flow rate of 0.5 mL/min.19 2.6. Methylation analysis Methylation was performed according to the method we previously reported.12,19 Briefly, the permethylated samples were hydrolyzed with 1.0 mL TFA (2.0 mol/L) at 110 °C for 6 h, and the hydrolysates were converted to alditol acetate before analyzing with GC–MS on a DB-225 column. 2.7. NMR analysis 20 mg polysaccharide was dissolved in 1.0 mL of 99% D2O, followed by centrifugation and lyophilization. The process was repeated twice and the final sample was dissolved in 0.5 mL of 99.98% D2O with one drop of acetone as internal standard (2.225 ppm for 1H and 31.07 ppm for 13C). The 1D 1H and 13C NMR, 2D 1H–1H correlated spectroscopy (COSY), 1H–13C heteronuclear multiple quantum coherence (HMQC), and 1H–13C heteronuclear multiple-bond correlation (HMBC) were recorded on JEOL JNM-ECP 600 spectrometers at 23 °C.14 3. Results and discussion The yield of FO1 was about 0.12 g/L, and gel permeation chromatography gave a symmetrical peak with an average molecular weight of about 36.0 kDa (Fig. 1). The carbohydrate content of FO1 was 98.9 based on the phenol–H2SO4 method. It had a negative response to the Bradford method, Therho and Hartiala reaction, and carbazole–H2SO4 method, suggesting the absence of protein, sulfate ester, and uronic acid, respectively. GC analysis for neutral sugars gave galactose and glucose in 50.6% and 49.4%, respectively. Methylation analysis gave two major partially methylated alditol acetates, 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-glucitol and 1,2,4-tri-O-acetyl-3,5,6-tri-O-methyl-D-galactitol in a peak area ratio of 1.02:1.00. Therefore, this exopolysaccharide may contain disaccharides as the repeating unit. The galactose residue of this repeating unit was furanoid and the glucose residue was pyranoid. These results were confirmed by NMR spectroscopy. In the anomeric region of 1H NMR spectrum (Fig. 2a), two signals appeared at d 5.03 and 4.93 ppm with integrated peak areas 1.03:1.00, which were labeled A and B from low to high field, and this result was in reasonable agreement with the results of chemical analysis. About 12 major peaks appeared at the high field, all of which should be assigned to H2–H6 of units A and B. In the 13 C NMR spectrum (Fig. 2b), two anomeric carbon signals appeared at 107.11 and 98.84 ppm. The former is derived from the b-D-Galf residues according to its chemical shift, since the anomeric carbon signals of a- and b-galactopyranose and a-galactofuranose do not exceed d105 ppm.20 A lower field signal than d 105 ppm is only observed for the b-configuration of galactofuranoside. The anomeric

Figure 1. The standard curve of dextran and the HPGPC chromatogram of FO1. (a) The standard curve of dextran with molecular weights of 788, 404, 112, 47.3, 22.8, 11.8, and 5.9 kDa. (b) The HPGPC chromatogram of FO1. HPGPC is short for high performance gel permeation chromatography.

carbon signal at 98.84 ppm is derived from the a-D-Glcp residues according to our previous publication.21 Two negative peaks were observed at d 67.67 and 61.39 ppm in the DEPT spectrum (Data not shown), which reflect that C-6 of the residues and the former may be assigned to the 6-O-substituted Glcp. Based on a series of 1H–1H COSY, and 2D 1H–13C (HMQC and HMBC) 2D correlation experiments, we could assign the chemical shifts of 1H and 13C of the residues and determine the structure of FO1 unambiguously. The proton and chemical shifts values are shown in Table 1. From 1H,1H-COSY spectrum, all of the correlation signals could be assigned. As labeled in Figure 3a, the ring protons of unit A H1 (5.03 ppm) ? H2 (4.03 ppm) ? H3 (4.10 ppm) ? H4 (3.88 ppm) ? H5 (3.85 ppm) ? H6a (3.77 ppm) and H6b (3.63 ppm), and the ring protons of unit B H1 (4.93 ppm) ? H2 (3.43 ppm) ? H3 (3.57 ppm) ? H4 (3.29 ppm) ? H5 (3.61 ppm) ? H6a (3.72 ppm) and H6b (3.51 ppm) were determined. The proton– carbon correlation was assigned based on the HMQC spectrum (Fig. 3b), which maps the connectivities between carbon atoms and their directly bonded protons. For unit A, the correlations of H1 with C1 at 107.11 ppm and H2 with C2 at 87.42 ppm indicated this unit belongs to the [?2)-b-D-Galf(?]. For unit B, the relative signals arose from H1 with C1 at 98.84 ppm and H6 (a, b) with substituted C6 at 67.67 ppm were observed. The rest of the proton and carbon crosspeaks could also be assigned clearly by HMQC spectrum, all of which demonstrated that unit B belongs to the [?6)-a-D-Glcp(1?]. To further determine the structure of FO1, HMBC spectrum was recorded as shown in Figure 3c. Such spectrum shows long-range connectivities between carbon atoms and their coupled protons through two or three bonds. Some useful coupling signals through a glycosidic linkage could be detected with certainty. The glycosidic connectivities of FO1 were determined by the presence of cross-peaks between H1 of unit B

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Figure 2. The 600-MHz 1H and 13C NMR spectra of FO1 recorded on JEOL JNM-ECP 600 Hz spectrometer in D2O at 23 °C. (a) 1H NMR spectrum of FO1. (b) 13C NMR spectrum of FO1.

Table 1 The NMR signals assignment of the exopolysaccharide FO1 produced by Fusarium oxysporum Y 24-2 Linkage pattern

?2)-b-DGalf(? A ?6)-a-D-Glc(1? B

Chemical shift:

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C/1H (ppm, 10-6)

1

2

3

4

5

6(a)

6(b)

107.11 5.03 98.84 4.93

87.42 4.03 71.96 3.43

76.26 4.10 73.62 3.57

83.25 3.88 70.36 3.29

70.36 3.85 73.16 3.61

61.39 3.77 3.63 67.67 3.72 3.51

and C2 of unit A. Thus, unit B is linked to C2 of unit A unequivocally, that is to say, FO1 is composed of galactofuranose residues substituted at C2 and glucopyranose residues substituted at C6 alternately. Additionally, some correlation signals within the residues were observed, such as A H1/C5, A H3/C2, A H4/C3, B H1/C5, B H2/C3, B H4/C5, B H6a/C4, and B H3/C5. Furthermore, the intraring cross-peaks A H1/ C4 and B H1/C5 demonstrated the furanoid character of ring A and the pyranoid structure of ring B. The above connectivities and substitutions are in agreement with the chemical analysis results and allow us to propose the structure of this exopolysaccharide FO1 produced by Fusarium oxysporum unequivocally as following (n  111):

Most of the polysaccharides from microorganisms containing galactofuranose are galactomannans, their structural differences mainly arise from the degree and sequence of the side chains of

(1?2), (1?3), (1?5), and (1?6) linked b-D-galactofuranosyl units,22,23 and all of the side chains are linked to the mannan core generally consisted of (1?2) or (1?6) linked a-Manp units. Results indicated the structure of the exopolysaccharide FO1 differs from the cell wall polysaccharide of the Fusarium oxysporum reported previously,10,11 as the cell wall polysaccharide is described to be composed of glucosamine and N-acetylglucosamine (25–35%). FO1 also differs from the cell wall polysaccharide of Fusarium sp. M7-1, which consisted of mannose, glucose, galactose, and glucuronic acid.5–7 However, a small quantity of [?2)-b-DGalf(1?] and [?6)-a-D-Glcp(1?] glycosyls present in the cell wall of Fusarium sp. M7-1 as oligosaccharides8 and [?6)-a-D-Glcp(1?] glycosyls also present in other Fusarium strains as investigated by FT-IR spectroscopy.2 Alkali-extractable and water-soluble cell-wall polysaccharides of some species of Fusarium consisted of a main chain of b-(1?6)-linked galactofuranose units almost fully branched at positions O-2 by single residues of glucopyranose or acidic chains containing glucuronic acid and mannose as determined by chemical analysis and NMR. To the best of our knowledge, this is the first time to report a polysaccharide with [?2)-b-DGalf(1?6)-a-D-Glcp(1?] units produced by Fusarium oxysporum. Carbohydrate produced by endophytic fungi is found to influence the metabolites of its host. For example, the exo- and cell wall polysaccharides and the oligosaccharides with different degrees of polymerization produced by the endophytic fungus (Fusarium oxysporum Dzf17) isolated from the rhizomes of Dioscorea zingiberensis were found to significantly enhance diosgenin accumulation in Dioscorea zingiberensis cell cultures.24,25 The function of FO1 is currently under investigation in our laboratory. Furthermore, as polysaccharides are considered as useful characters for the grouping

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Figure 3. The 600-MHz two-dimensional NMR spectra of FO1 recorded on JEOL JNM-ECP 600 Hz spectrometer in D2O at 23 °C. (a) 1H–1H COSY spectrum of FO1. (b) HMQC spectrum of FO1. (c) HMBC spectrum of FO1.

of microorganism and the proposal of evolutionary theories,3 therefore, the structural elucidation of this novel polysaccharide FO1 may be helpful to the classification and evolutionary study of the fungus Fusarium species. 4. Conclusions The structure of the exopolysaccharide FO1 produced by Fusarium oxysporum Y24-2 consists of [?2)-b-DGalf(1?6)-a-D-Glcp(1?] units, which differs from the previously reported polysaccharides, including the cell wall polysaccharides from the mycelia of Fusarium species. Acknowledgments This work was supported by Grants from the Science and Technology Development Program of Shandong Province, China (2010GHY10509), and Grants (1065 and 2234) from Taishan Medical University. This work was also supported by Taishan Scholars Foundation of Shandong Province (China). References 1. Yera, H.; Bougnoux, M. E.; Jeanrot, C.; Baixench, M. T.; De Pinieux, G.; DupouyCamet, J. J. Clin. Microbiol. 2003, 41(4), 1805–1808.

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