Indian journal of Experimental Biology Vol. 43, May2005, pp. 467-47 J

Characterization of an exopolysaccharide produced by a marine Enterobacter cloacae

Anita lyeI', Kalpana Mody* & Bhavanath Jha

Central Salt and Marine Chemicals Research Institute, G B Marg, Bhavnagar, 364 002, India

Received 24 November 2004; revised 23 Jalluary 2005

An exopolysaccharfde producing marine bacterium, £lIterobacter cloacae, was isolated from marine sediment collected from Gujarat coast, India. Chemical investigation of exopolysacclmride (EPS 71 a) revealed that this exopolysaccharide was an acidic polysaccharide containing high amount of monic acid, fucose and sulfate which is rare for bacterial exopoly-saccharides. EPS 71a was found to have fucose, galactose, glucose and glucuronic acid in a molar ratio of 2 : 1 : 1 : I.

KeywordS: Bacteria, £lIterobacter cloacae, Exopolysaccharides, Fucose, Galactose, Glucose, Glucuronic acid , Marine bacteria

Microbial cells generally contain various polysaccharide structures contributing to their shape and rigidity. These polysaccharides are of three types and have different functions in a microbial cell-(a) intracellular polysaccharides that provide mechanisms for storing carbon or energy for the cell ; (b) structural polysaccharides that are components of the cell structures such as lipopolysaccharides and teichoic acids present as integral components of cell walls; and (c) extracellular polysaccharides referred to as exopolysaccharides (EPS). Exopolysaccharides produced by a wide variety of microorganisms are water-soluble gums having novel and unique physical

. I properties .

The structure of polysaccharides is relatively simple, comprising of homopolysaccharides (polymers containing one type of sugar) or heteropolysaccharides (containing more than two types of monosaccharide units). Many polysaccharides contain acyl groups as additional adornments. The commonest acyl substituents are acetate esters and pyruvate ketals; succinyl half esters are also featured in exopolysaccharides. The presence of ketals or uronic acids results in linear polyanionic macromolecules. Some polysaccharides have larger repeating units and other acyl substituents . These include succinoglycan produced by RhizobiulIl and AgrobacteriulIl spp., which is composed of D-glucose and D-galactose in an octasaccharide-repeating unit

'Correspondent author Phone: 0278-2561354 Fax: 027X- 2566970 Email: khmody@csll1cri.org

and carries O-acetyl groups, O-succinyl half-esters and pyruvate ketals2.

The chemical nature of exopolysaccharide plays a vital role in its property3. It has been widely accepted that carboxylic acids and amide groups are mainly responsible for the metal binding capacity .of exopolysaccharide. Hydroxyl groups are probably involved in the chelation of metallic ions as oxygen atoms could be weak electron donors . . Such complexes of metal ions with neutral carbohydrates are well documented by Angyal4 . The presence of .

hydroxyl group in ~-position can contribute to the stability of such complexes . Alduronate ions form much stronger complexes with cations than neutral sugars. Kaplan et al. s have reported that the sulfate ester group plays a minor role in metal chelation as compared to uronic acid.

Iyer et al. 6.7 reported bioacculllulation of heavy metals by an exopolysaccharide (EPS 71 a) produced by a marine bacterium, Elllerobacter cloacae. In continuation of this, the present study incLudes chemical investigation of EPS 71 a, which w·ould highlight its added applications.

Materials and Methods An exopolysaccharide producing bacterial culture

was isolated frolll a marine sediment sample collected from Gujarat coast, West coast of India. [t was identified using the API system (Amin's Laboratory , Baroda, India) and confirmed by using biochemical tests. Zobell Marine Agar2216 (HiMedia M 384) was used for maintenance of this culture. For production of exopolysaccharide, the culture was grown in

468 INDIAN J EXP BIOL, MAY 2005

Seawater brothS containing (g/I seawater) sucrose, 30.0; peptone, 5.0; yeast extract, 1.0; and agar, 30.0 and incubated at 2r-30°C for 76 hr. The broth containing the grown cells was centrifuged and the supernatant was precipitated in 3 volumes of isopropanol. The precipitates were dried, dialyzed and lyophilized to obtain crude exopolysaccharide l . The exopolysaccharide product thus obtained was named EPS 71a.

EPS 71a was analyzed for its total sugar, sulfate, protein and uronic acid contents using the standard methods9• 12 .

A.nion exchange chromatography of EPS 71a was carried out on a DEAE cellulose column (CI" form) (Sisco Research Laboratories Pvt. Ltd., India) (45 x 3 cm), which was eluted using a step-wise gradient of 0.0 to 2.0 M aqueous sodium chloride (NaCl) solution at 25°C at a flow rate of 3 mllmin . One gram of EPS 71a, dissolved in minimum amount of water, was charged to the anion exchange chromatography column. Fractions were eluted with distilled water followed by stepwise gradient of sodium chloride solution (0.5, 1.0 and 2.0 M). The fraction size was 25 ml and they were monitored for the presence of sugar using phenol-sulphuric acid method9. Fractions rich in sugar (exopolysaccharide-enriched) were pooled, dialyzed over-night against distilled water and subsequently lyophilized. The fraction yielding maximum product was subjected to monosaccharide analysis using gas liquid chromatography (GLC).

Crude product as well as DEAE cellulose column chromatographic fraction of EPS 71a obtained usin o b 0.5 M of NaCI solution were subjected to monosaccharide composition analysis using gas liquid chromatography (GLC). Glycosyl composition of the exopolysaccharide was determined by methanolysis using methanolic HC!. The methyl glycosides were converted to the corresponding trimethylsilyl derivatives as described by Montreuil et al.l3 . Analytical GLC was performed on a Fisons instruments GC 8000 series fitted with a WCOT fused silica CP-SIL 5CB (60 m x 0.25 mm) with a temperature program of 50-120°C at 20 °C/min, then 120-240 °C at 2°C/min and hydrogen as carrier gas (Courtesy: Prof. Jean Guezennec, Head of Biotechnology Department, IFREMER, France). The lype and percentage of different monosaccharides were determined by GLC using erythritol as the internal reference. The absolute configuration of sugars was determined by comparison of GLC

analysis of their corresponding standard trimethylsilylated R( -)-2-butyl derivatives 14.

Infra red spectra of crude EPS 71 a was recorded in potassium bromide pellets using FT-IR spectrophotometer (Perkin- Elmer Spectrum GX).

Results API system was used in the present investigation

for identification of the bacterium. This system identifies bacterial cultures based on carbohydrate utilization. Additionally, biochemical tests were also carried out to support the identification. Based on both the methods the bacterium in study was identified as Enterobacter cloacae.

As shown in Table 1, crude EPS 71 a contained (%) 45, total sugar; 18.75, protein; 7.0, sulfate; and 9.23, uronic acid, which amounted to approximately 80%. The remaining 20% may be accounted as impurities other than exopolysaccharides. The DEAE cellulose column chromatographic fractionation of EPS 71a' yielded only 20 % recovery with maximum yield of 9.4 % with 0.5 M of sodium chloride solution followed by 1.0 M of NaCl (4.2 %) and 2.0 M of NaCI solution (2.6 %; Table 2). The distilled water fraction of this exopolysaccharide yielded 2.5 % product. Each of the NaCI fraction was individually analyzed for protein, total sugar, uronic acid and sulfate contents and compared with those of crude EPS 71 a (Table 2). The 0.5 M NaCl fraction was subjected to further analysis as it yielded the maximum product.

Total sugar content of crude EPS 71 a was 45 %, whereas it ranged from 11-55.7 % in the column fractions . Maximum sugar content was observed in

Table 1- Yield and chemical analysis of EPS 71 a

Yield (%) 0.51

Uronic acid (%)

Sulfate (%)

Protein (%)

Total Sugar (%)

9.23

7.0

18.75

45.16

Table 2-Yield and chemical analysis of DEAE cellulose column chromatographic fractions of EPS 71 a

Fractions Yield Sugar Sulfate Protein Uroni c (%) (%) (%) (%) acid (%)

0.5 M 9.4 55.7 3.7 6.4 16.7 Nael

1.0 M Nnel

2.0M Nael

4.2 32

2.6 II

3.63 10 14

6.87 9 7

IYER et al.: CHARACTERIZATION OF AN EXOPOLYSACCHARIDE PRODUCED 469

37.0 71a 36 1-3-1000

34

32

30

I

2' I I I

37S3. ,

26

~

~ 24

22

20

1.

16

14

13.0

4000.0

\ \ \ I \ \

2921.32

3000

---/ I I I

I I I I

2370.n I I I I

/

2170.10

r'"

2000

I I I I I

1623.93

" I 140(.7. : :

I I I 12H.20 I I

I "

f072 S8

13".30 1149.n

uoo 1000 soo 400.0 em":' l

Fig.I-IR spectra of EPS 71 a

Table 3-- Monosaccharide composition determined using GLC

Constituent sugar (%)

Sample

EPS 71a

Rhamnose Mannose Fucose Galactose Glucose Glucuronic acid

·0.5 M NaCI fraction

0.8

1.4 0.3

0.5 M NaCI fracti'on (55.7 %) which also contained high uronic acid (16.7%) and low protein (6.4 %) and sulphate (3.7%) contents.

Gas liquid chromatographic analysis of EPS 71a and its 0.5M NaCI fraction revealed the presence of unusually high amount of fucose (25-26%), which is quite rare for bacterial exopolysaccharides. It also contained galactose, glucose and glucuronic acid. The molar ratio of the monosaccharide i.e. fucose, galactose, glucose and glucuronic acid present in EPS 71 a can be expressed as 2 : I : 1 : 1 (Table 3).

lR spectrum of EPS 71 a (Fig. 1) exhibited a broad O-H stretching absorption band at around 3400 cm'l and a weak adsorption band at 2930-2928 cm,l, which was attributed to C-H. The presence of C-H revealed a good reference to total sugar content. Significant band of -COOH groups (1722 cm'l) was also

25 .6

26

11.1

13.7

1l.6

1l.7

9.4

9.7

observed. A strong absorption band at 1623 cm'l was assigned to the stretching vibration of carboxyl group. These carboxyl groups present on exopolysaccharide can serve as a binding site for divalent cations. A band of amide from protein could also be revealed at 1408 cm'l. A strong band at 1253 cm'l indicated the presence of S=O of ester sulfate, whereas the strong stretching band at 1072 cm'l was assigned to C-O.

Discussion Non-sugar components, including sulfate and

protein make up a relatively smaller portion of exopolysaccharide on a per weight basis. However, they may be extremely important to the tertiary structure and physical properties of the exopolysaccharide. These components are most often in the form of side-groups on the polysaccharide

470 INDIAN J EXP BIOL, MAY 2005

chain as carboxyl and sulfate groups. The occurrence of these non-sugar components imparts acidic nature to the polymer. The presence of high uronic acid confers -an· overall negati ve surface charge and acidic properties to the exopolysaccharide l5 . Such negatively charged surfaces of polysaccharides may play an important role in their metal compJexing capacity. It has been reported that exopolysaccharide such as algi nates produced by Azotobacter villelandii and P d . 16 h' h . I sell 0111 alias aeruglilOsa , w IC are negatIve y charged and rich in uronic acid, exhibit a higher metal chelating capacity. Such exopolysaccharides have been reported to exhibit high copper binding capacity which widens their application in the field of biodetoxification and wastewater treatment3• 17-1 8.

EPS 71 a has approximately 9% uronic acid. Similarly , almost the same amount of -uronic acid contallllng polymer B of an extracellular polysaccharide from a marine Vibrio has been reportedl~. However, it varies in its monosaccharide composition i.e. galactose:glucose (I: 13). Another xanthan like bacterial exopolysaccharide produced by Elllerobacler agglollleralls is also reported to contain uronic acid (38.9%). This polysaccharide stabilizes emulsions requiring low viscosit/o.

In our earlier studies, EPS 71 a was found to exhibit promising heavy metal chelating propert/,·7 which might be correlated to its high uronic acid content. Also, this may offer some selective ecological advantage to this bacterial culture, as it may help the culture to grow on surfaces coated with toxic and antifouling compounds such as cuprous oxide.

Antiviral activity of sulfated derivatives of a fucosam~ne-containing polysaccharide of a marine Pseudolllollas sp. has been I'eported by Okutani21. Though, this native polysaccharide did not contain any -sulfate group, its column fractions contained 0.08-0.79 sulfate groups per sugar residue. Thes.! sulfate values are lower as compared to other sulfate,i polysaccharides with antiviral activity such as heparin (1 .25 sulfate group per sugar resid ue) and dextran sulfate (2-3 sulfate groups per sugar residue). Although , the antiviral effects appear to require a high degree of sulfation of the molecule, exopolysaccharide from Pseudolllollas sp. exhibited anti HSV -I effect. I n the present studies, EPS 71 a contained 7 % of sulfate, which may be inadequate to exhibit antiviral activity . However, this may be confirmed after carrying out bioassay for antiviral activity. Inhibitory effects of various polyanions on

viruses have suggested that these substances influence the primary electrostatic attachment of the virus to the cell prior to viral penetration22.

Sulfated polysaccharides isolated from CodiulIl tomentoSlll1l and CodiulII dwarkellse have been reported to exhibit heparin-like blood anticoagulant

. . 23-25 M f If . actIvIty . oreover, presence 0 su ate 111 exopolysaccharide may play an important role in protection, desiccation and cation exchange property of a bacterial biofilm. Majumdar et al. 26 have studied an exopolysaccharide producing Vibrio spp. that was isolated from a biofilm material. This exopolysaccharide exhibited anticorrosive property for mild steel.

Certain microbial polysaccharides, best known for their thickening, gelling or emulsifying properties are easy sources of unusual 6-deoxyhexose i.e. L-fucose. L- Fucose can be used as a substrate in the chemical synthesis process of furanone-flavoring compounds. L-Fucose production via chemical synthesis is laborious and suffers from low yield, while direct extraction from brown algae is costly and subject to seasonal variations in quantity and quality. Chemical or enzymatic hydrolysis of L-fucose rich microbial exopolysaccharide opens up a new route towards efficient L-fucose production. Vanhooren and Vandamme27 have reported microbial production of clavan by Clavibacler lIIichigallellsis as a source of L-fucose. A similar type of exopolysaccharide, produced by Klebsiella plleulllolliae contalllll1g L-fucose (18 .9 % w/w) has been reported as a source of L-fucose:!7. This exopolysaccharide has been commercialized under the trademark Fucogel and produced industrially in a complex medium. Production of fucogel, L-fucose rich exopolysaccharide of Klebsiella pllelllllolliae has been studied by Ramirez-Castillo et 0/28 . EPS 71 a contains unusually high amount of fucose (25-26 %) and therefore, it can be exploited as a source of fucose .

Furthermore, L-fucose and L-fucose containing oligosaccharides have potential application in the medical field in preventing tUlnor cell colonization of the lung (anticancer effect), in controlling the formation of white blood cells (anti-intlamm

IYER et al.: CHARACTERIZATION OF AN EXOPOLYSACCHARTDE PRODUCED 471

bioactivity of this exopolysaccharide to prove its application in medical field.

Acknowledgement The authors are grateful to Dr P K Ghosh, Director,

CSMCRI, Bhavnagar for encouragement. One of the authors (AI) is thankful to CSlR for financial assistance.

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