A special levan-type exopolysaccharide (EPS) from Bacillus amyloliquefaciens JN4 with antiadhesive activity against enterotoxigenic Escherichia coli (ETEC) was purified and identified. Chemical analysis indicated that EPS-JN4 with a low molecular weight of 8 kDa is composed of fructose and glucose with a molar ratio of 46.1:1. Structural analysis clarified that EPS-JN4 contains a main chain of β-(2,6)-linked Fruf residues and intensive branches of a single 2-linked Fruf at every six residues. Furthermore, the superior antiadhesive activity of EPS-JN4 against ETEC showed its potential usage as an antiadhesive agent for diarrhea prevention. EPS-JN4 is a specific type of levan family, for its small molecular size and intensive branches. The results expand the knowledge on structural types of levan and illustrate its potential as an antiadhesive agent for diarrhea prevention, which will be conducive to elucidate the relation between structure and function.

enterotoxigenic, exopolysaccharide, enterotoxigenic Escherichia coli, levan, antiadhesive, Bacillus amyloliquefaciens

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6	bDFruf	62.4	106.7	78.8	77.7	82.9	66.0
6,6,1	bDFruf	62.7	106.3	78.8	77.7	83.6	64.7
6,6	bDFruf	62.4	106.0	79.2	76.8	82.9	65.8
	bDFruf


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6	bDFruf	3.62-3.74	-	4.16	4.07	3.89	3.49-3.87
6,6,1	bDFruf	3.64-3.74	-	4.14	4.07	3.84	3.84
6,6	bDFruf	3.65-3.71	-	4.20	4.07	3.93	3.52-3.83
	bDFruf


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6	bDFruf	62.4/3.62-3.74		78.8/4.16	77.7/4.07	82.9/3.89	66.0/3.49-3.87
6,6,1	bDFruf	62.7/3.64-3.74		78.8/4.14	77.7/4.07	83.6/3.84	64.7/3.84
6,6	bDFruf	62.4/3.65-3.71		79.2/4.20	76.8/4.07	82.9/3.93	65.8/3.52-3.83
	bDFruf

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]OC[C@H]1O[C@@](CO[C@]2(CO)O[C@H](CO)[C@@H](O)[C@@H]2O)(OC[C@H]2O[C@@](CO)(OC[C@H]3O[C@@](CO)(OC[C@H]4O[C@@](CO)(OC[C@H]5O[C@@](CO)(OC[C@H]6O[C@]([*])(CO)[C@@H](O)[C@@H]6O)[C@@H](O)[C@@H]5O)[C@@H](O)[C@@H]4O)[C@@H](O)[C@@H]3O)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O

1134.987 g/mol (C₄₂H₇₀R₂O₃₅, R = next and previous repeats, not counted in mol weight)

In this study, a bacterium with the ability to extracellularly produce a water-soluble polysaccharide (with high molecular mass of 743 kDa) was obtained from saline soils. This strain named as ZB01 was identified as Rhizobium punsense by 16S rRNA sequence analysis. The monomer composition and structure of extracellular polysaccharides were investigated by high-performance anion-exchange chromatography-pulsed amperometric detector, Fourier-transform infrared, methylation and nuclear magnetic resonance spectroscopy analysis. In addition, the morphological characterization of extracellular polysaccharides was performed by scanning electron microscopy analysis. As a result, the polysaccharide is found to be a novel water-soluble type of glucan most likely consisting of repeating two β-D-Glcp-(1→3) and one α-D-Glcp-(1→3). Furthermore, it showed pronounced antimicrobial effects against Escherichia coli and Bacillus subtilis, suggesting it has the potential to be used as a natural antibacterial agent in the future.

structure, exopolysaccharide, glucan, antimicrobial activity, Rhizobium pusense

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3	bDGlcp	103.72	74.42	84.10	69.32	78.42	63.92
3	aDGlcp	?	72.97	81.28	70.87	74.37	60.99
	bDGlcp	102.53	75.47	84.06	69.50	78.46	62.36


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3	bDGlcp	4.50	3.52	3.62	3.34	3.53	3.56
3	aDGlcp	5.26	3.12	3.68	3.50	3.41	3.60
	bDGlcp	4.40	3.27	3.52	3.21	3.46	3.66


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3	bDGlcp	103.72/4.50	74.42/3.52	84.10/3.62	69.32/3.34	78.42/3.53	63.92/3.56
3	aDGlcp	?/5.26	72.97/3.12	81.28/3.68	70.87/3.50	74.37/3.41	60.99/3.60
	bDGlcp	102.53/4.40	75.47/3.27	84.06/3.52	69.50/3.21	78.46/3.46	62.36/3.66

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O[C@H]2[C@H](O)[C@@H](CO)O[C@H](O[C@@H]3[C@@H](O)[C@H]([*])O[C@H](CO)[C@H]3O)[C@@H]2O)[C@@H]1O

486.423 g/mol (C₁₈H₃₀R₂O₁₅, R = next and previous repeats, not counted in mol weight)