Biopolymers return to aqueous state again (Harada et al.,

Biopolymers are gaining more importance in our day-to-day life, due to the hazardous effect of synthetic plastics in our environment. Curdlan is one of the biopolymers, which has a broad field of applications in the food, pharmaceutical and medical. It is a polymer of glucose, produced by the family members of Rhizobiaceae.  Curdlan (? (1,3) d glucan) production takes place under the nitrogen starvation period during the cell growth. Fifty Agrobacterium sp. were isolated from root nodules collected from different localities of Coimbatore and Palakkad and screened by Aniline Blue method for the production of curdlan. The species was confirmed as Agrobacterium fabrum by 16S rRNA sequencing. The accession number given by GenBank is MF521602. Curdlan was produced using different carbon and nitrogen sources. The extraction was done by Sodium hydroxide: Acetic acid precipitation. 0.12 gm of crude Curdlan was obtained from 1.4 gm/100 ml of biomass. The product was stored for further research.Keywords:   Biopolymer, Curdlan, 16S rRNA, GenBank accession, Agrobacterium fabrum, Sodium hydroxide: Acetic acid precipitation. Exopolysaccharides are the secondary metabolites produced by a vast group of microorganisms. One such group of organisms which produce these types of polysaccharides is the Rhizobiaceae family. The genus Agrobacterium has the nature of producing a special group of polysaccharides (1?3)- ?-D- Glucan, termed as Curdlan. These nitrogen-fixing root nodule bacteria have the ability to produce these special polysaccharides in the nitrogen starvation conditions. Gelling and emulsifying nature of curdlan has gained application in the industrial sectors (Sutherland 2001). Xanthan, Gellan, and Curdlan are the three exopolysaccharides, which has been approved as Food additives by the Food and Drug Administration (Jezequel 1998). Curdlan clumps as a strong gel, in the thermal suspension totally, never return to aqueous state again (Harada et al., 1968).Structural analysis of curdlan is given as ?-(1?3) linkage with a homopolymer of D-glucan (Saito et al., 1968). The degree of polymerization (DP) of unbranched curdlan was roughly measured as 450 (Nakanishi et al., 1976) It has a major impact in clinical level, as it can elicit the immune response against many pathogenic microbes and drastic disorders like cancer ( Harada et al., 1993).Large-scale production of exopolysaccharide curdlan is performed mainly by Agrobacterium sp. ATCC 31749 (Anne M Ruffing and Rachel Ruizhen Chen, 2012). Curdlan biosynthesis requires the participation of four major genes crdA, crdS, crdC and crdR (Stasinopoulos et al., 1999). NtrB and NtrC molecules give a hand for the synthesis of curdlan under nitrogen retardation period (Mcintosh M et al., 2005).MATERIALS AND METHODS:1. Sample collection, Isolation of Agrobacterium sp and Screening for the curdlan production: Root nodules were collected from the different locality of Coimbatore and Palakkad carefully without disturbing the nodules and preserved in the carry bags routinely. Root nodules were washed under running tap water to remove the excess soil, and surface sterilized with 1% mercuric chloride solution (Sharma et al., 2005). Sterilized nodules were carefully handled with sterile forceps and pressed under the two sterilized glass slide to extract the fluid inside the nodules. The extract was streaked on the sterile Yeast extract mannitol agar plates, with sterilized loops (Vincent, 1970). Isolated organisms were inoculated into a Yeast extract mannitol agar containing Aniline Blue reagent (Nakanishi et al., 1974).2. Characterization of Positive Agrobacterium Isolates: Agrobacterium isolates characterized by Congo red test (Hann, 1966), growth on 3-Ketolactose test, Citrate utilization test, Hofer’s alkaline broth (Hofer, 1941), and growth in glucose peptone agar (Subba Rao, 1981). The organism was confirmed by 16S rRNA sequencing technique done in Yaazh Xenomics lab at Coimbatore.3. Seed Culture preparation, production & Extraction of Curdlan: The inoculums for the production of curdlan was prepared by inoculating the isolates in the seed culture media containing peptone 0.5g, yeast extract 0.5g and 1% of sucrose as a carbon source. The medium was kept for incubation at room temperature for 24-48 hours. The seed culture inoculums were inoculated in the Minimal salt medium containing KH2P04, 3.0; Na2HP04, 6.0; MgS04, 0.5; (NH4)2HP04;0.1, trace element solution- 10ml (5g FeS04.7H20, 1g CoCl2.6H20, 2g MnS04, citrate, 1g ZnCl2 per litre of 0.1mol l-1 HCl). Medium adjusted to pH 5.5 (Lee et al., 1997c). The inoculated flasks were kept for shaking incubation at room temperature for 76 hours at 180rpm. The extraction of curdlan  (Lee et al., 1997a, Lee et al .,1997b), done by centrifuging the culture medium at 12,000×g for 30 min. The extracted pellet was mixed with the same ratio of 0.5N Sodium hydroxide at 4?C. The solution was kept undisturbed for 3 hours at the same temperature. After that the content was again centrifuged at 12,000 ×g for 40 min, supernatant obtained after centrifugation contains the Curdlan. This was precipitated by adding 10% acetic acid and was twice washed with water, acetone, and ether. 1. Collection, Isolation & screening: Root nodules collected from the regions of Coimbatore and Palakkad. Fifty Agrobacterium samples were isolated from the root nodules collected from different regions of Coimbatore and Palakkad.  Red coloured mucoid colonies were obtained on Yeast Extract mannitol agar plates. The fifty samples were screened by Aniline blue method. 7 of the samples showed positive result by producing blue color colonies.2. Characterization of Agrobacterium sp: The samples showed red colour colonies on the congo red test. The samples produced yellow color on the lactose agar as a positive result. On Hofer’s alkaline broth and glucose peptone agar, the samples showed maximum growth. The samples were positive for Citrate utilization test. The organism was found to be Agrobacterium fabrum by 16S rRNA sequencing. The accession number given by GenBank is MF521602.3. Seed culture preparation, production &Extraction of curdlan: The 5 samples showed high cell growth in the seed culture medium. A high dense growth of organism was observed in the production medium, pH 5.5 was reported as the optimal for curdlan production. Crude curdlan was extracted by the centrifugation method, 1:7.5 or increased ratio level between samples to alkali given by Lee et al. 1999 for the extraction of curdlan.