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Journal of Life Sciences and Technologies Vol. 1, No.

1, March 2013

Synthesis and Characterization of Cyclic β-(1,
2)-Glucan from Agrobacterium Tumefaciens
Geetha Venkatachalam and Mukesh Doble
Bioengineering and Drug design Lab, Department of Biotechnology, Indian Institute of Technology Madras, India
E-mail: geegha@gmail.com, mukeshd@iitm.ac.in

Abstract—Biodegradable polymers are used as carriers for
controlled delivery of low molecular weight drugs as well as A. Experimental Section
bioactive proteins. Synthesis and characterization of a Optimization of culture conditions for cyclic β-(1,
biopolymer, cyclic β-(1, 2)-glucan from Agrobacterium 2)-glucans production by Agrobacterium tumefaciens was
tumefaciens is reported here. The culture condition for its carried out in shake flask condition. Growth of the bacteria
production in the extracellular medium in shake flask was determined in Luria broth at different time intervals.
condition is optimized. Maximum biomass production is
They are incubated at 37°C for 60hr and the OD was
obtained at 32hr in Luria broth and glucan production is
observed at 48hr in mannitol media. The polymer is purified measured at 600nm with UV-Visible spectrophotometer.
by Biogel P6 column chromatography and characterized by Three different mediums were used for cyclic glucan
TLC, 1H NMR, FT-IR analysis.  production namely PM-1 (Breedveld et al., 1995) which
contains (g/l) Yeast extract-1.0g, Glucose-5.0g, K2HPO4 -
0.6g, MgSO4 - 0.01g, FeCl3 - Trace, KH2PO4 - 0.1g,
Keywords—Agrobacterium tumefaciens, cyclic β-(1, 2)-glucan,
1
H NMR, production CaCO3 - 0.5g, Glutamic acid - 0.1g, pH-7.5, and PM-2
medium contains, Yeast extract - 1g, Mannitol - 5.0g,
K2HPO4 - 1.0g, MgSO4 - 0.4g, FeCl3 -Trace, KH2PO4 -
I. INTRODUCTION 0.1g, Thiamine - 5mg, Biotin - 2µg, pH-7.5, and PM – 3
which contains Glucose -4.0g, (NH4)2HPO4 - 0.15g,
A majority of drug delivery systems using natural MgSO4 - 0.05g, FeCl3 - 0.001g, CuSO4.5H20- 5µg, Yeast
polymers are based on polysaccharides (e.g., starch, extract -1.0g, KH2PO4 - 0.1g, CaCO3 - 0.5g, Thiamine -
dextran, hyaluronic acid, and chitosan). Applications of 20µg, Biotin - 2µg, pH-7.0.The 24hr old culture were
proteins to deliver drugs have been limited due to their inoculated into the production medium and incubated at
poor mechanical properties, low elasticity, possible 37°C for 48hrs. The culture filtrate was centrifuged at
occurrence of an antigenic response, and the high cost. On 12,000 rpm and the supernatant was precipitated with 10
the other hand, polysaccharides have attracted increasing volumes of ethanol. The solvent system used for thin layer
interest as the drug carriers because they are commercially chromatography is ethanol: butanol: water in the ratio of
available at low cost, are readily modified by simple 5:5:4. The TLC plates were sprayed using 5% (vol/vol)
chemical reactions for specific applications, and they H2SO4 in methanol. The crude samples were lyophilized
exhibit a broad range of physicochemical properties. and concentrated. Glucans were purified by Biogel P6
Cyclic glucans are mainly found in Proteobacteria and column using the solvent system 5% acetic acid in water
Gram-negative bacteria [1]-[2]. Glucans produced by with flow rate of 2ml/min.
Rhizobiaceae are substituted with nonsugar moieties
including sn-1-phosphoglycerol [3]-[4], succinate [5] or B. Characterization of the Purified Fraction
methylmalonate [6]. The capacity of the cyclic β-glucans The 1H Nuclear magnetic resonance was recorded on a
to form inclusion complexes suggest that these molecules Bruker Avance III 500MHz NMR spectroscopy using
may find industrial applications similar to those currently D2O as solvent. Infra red spectrum was obtained with the
being explored for the cyclodextrins. Also, cyclic β-(1, 2)- help of Fourier transform- infrared spectrophotometer
glucans are more soluble in water than the cyclodextrins (Perkin Elmer).
(approximately 250 and 18g/liter for cyclic β–(1, 2) –
glucans and γ - cyclodextrins, respectively [7]. This III. RESULTS
property may be of value for pharmaceutical or The maximum growth of the organism was observed at
food-processing applications, in which high 32nd hr in Luria broth (Fig. 1). Maximum glucan yield
concentrations of inclusion complexes may be required. (62.0 mg/L) was observed in PM-2 medium (Fig. 2)
followed by PM3 (45 mg/L) and PM-1 (50 mg/L) (Table.
II. METHODOLOGY I). The cell pellets were precipitated and the intracellular
glucan 42mg/gm of biomass were obtained. The TLC
Manuscript received September 20, 2012; revised December 25,
2012.

©2013 Engineering and Technology Publishing 44
doi: 10.12720/jolst.1.1.44-46

"Isolation and characterization of periplasmic cyclic beta-glucans of azorhizobium caulinodans. Okada." Journal of the Pharmaceutical Society of Japan. G. " BMB Reports. 4.350 .8 0. J.000–1. Benincasa. 1986. Geetha Venkatachalam was born in Salem on 07. (Chennai-600036. Hisamatsu. M.2)-D-glucans) by agrobacterium and rhizobium species. Lee. Ikeda. "Studies on inclusion complexes of non-steroidal anti-inflammatory agents with cyclosophoraose-A. Marco. pp. No. in FTIR spectrum indicates the β linkage of the cyclic "Characterisation of glycerophosphorylated cyclic-β-1. She holds B.06. [6] M. Komaniecka and A.93(s). A.6 1. groups. 1987.3. 901. 2)-D-glucans (cyclosophoraoses) produced by rhizobium meliloti and rhizobium trifolii. 2) glucan Figure 1. Acta cm-1 are due to O-H stretching hydrogen-bonded hydroxyl (BBA)-Biomembranes. 1 H NMR shows chemical shift values at δ 4. and S. and L. [3] M. Redmond. R. and B.4 0. March 2013 shows the presence of cyclic glucan as a single oval black color spot (Fig. 1. 61.3. 2) glucan cyclic β-(1. 42. [2] I. 106. and K. and K. T. [7] Y.6 0.405 (q.416 . P. vol. pp." Appl Environ Microbiol. 3. 2) 3 (mg/L) glucan (mg/gm 2 of cell mass) 1 0 PM-1 50 15 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 Time (hrs) PM-2 68 42 PM-3 45 20 Figure 2. and M. vol. Horiyama. 119-126. vol. Yamada. Applied Microbiology degree in the year 2003 from Periyar University. "High-performance liquid chromatography of cyclic cm-1. India. Batley. IIT-Madras for analytical help. T." Biochim.Sc Microbiology in the year 2001 and M. Rolfe. 1 H NMR spectrum of extracellular cyclic β. Geetha is a Women Scientist (2010-2013) in Drug design and Bioengineering lab. 4). 2) glucan of Biotechnology IIT Madras. P. Cho. O-acetylated cyclosophorans (cyclic b-(1.2 0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 Time(hrs) Figure 5. 5). 1987. β -(1." Figure 3. pp. 115–122. 4.(1.2 OD at 600nm 1 0. 393. Figures and Table 263-271. India. T. 1995. pp. pp. Benesi.2) glucan (mg/100 ml) 6 5 Production Extracellular cyclic Intracellular 4 medium β-(1.100 Zevenhuizen. pp. 3). Breedveld. The wave number at 1243cm-1 [4] M. Dr. Djordjevic.(1.851 "Effect of phosphate limitation on synthesis of periplasmic cyclic (solvent peak). 163. 1987 [5] M. vol. Miller. Jung. Higashiura. Cartoni. 2)-glucans. J= 2Hz) and beta-(1. Journal of Life Sciences and Technologies Vol. 240-247. Biophys. 2003.D in Applied Microbiology and Botany in the year 2009 from University of Madras. vol. REFERENCES [1] S. J. 2) GLUCAN FROM 7 DIFFERENT PRODUCTION MEDIUMS Cyclic β-(1. Phone Author’s formal photo 45 .Sc. Tamilnadu and Ph. PRODUCTION OF CYCLIC Β. 1. Koizumi. M. 2) glucan FEMS Microbiol Lett. L. 227. 2-glucans glucan (Fig.575 (bs).(1. "The production of acidic. vol. 3. The absorption bands at 3600 and 3200 from a fast-growing rhizobium species. Glycosidic bonds were observed at 1. "Periplasmic glucans isolated from proteobacteria. Thin layer chromatography of cyclic β." Carbohydr Res. Rizzo. FT-IR spectrum of extracellular cyclic β. J. Growth curve of Agrobacterium tumefaciens in luria broth TABLE I. W. vol. Choma. S. C. F." J. Dr. 2009.1981 in Tamilnadu. 1. S. E. Chromatogr . pp.324 (m) (Fig. Department Figure 4. July.4 1.(1. 263-269. Coccioli. Glucan production (PM-2) at different time intervals by Agrobacterium tumefaciens ACKNOWLEDGMENT The authors thank Sophisticated Analytical Instrument Facility. 769-775. 1045-1053.

and Mukesh Doble. V. He has previously biopolymer and its application as drug carriers. S. Elsevier. 2012.com). V. N. Bioreactors.” Chemical Engineering Progress. microorganisms . a Fellow of Royal society of biotechnological potential. cyclic glucan production.K. Madras. M. vol. Tech and M. 37-46. Ravishankar. 36-42. properties and applications. October 31. T. No. Tech Author’s formal briefs in Microbiology. March 2013 +91-044-5122. Geetha. Homogeneous catalysis : Mechnanisms & industrial applications.. Suryanarayanan. “Internal mycobiota of marine Engineers.ac. and Texas A&M. He is in the editorial board of the journal CHEMICAL macroalgae from the Tamilnadu coast: distribution. India and a Ph. 3.A. Thirunavukkarasu. diversity and ENGINEERING (McGraw Hill publn). He has authored “Fungal symbionts of marine sponges from Rameswaram. 53. Marcell Dekker. Doble. Geetha. Elsevier. UK and postdoctoral at Venkatachalam.” Fungal Chemistry and processes. DOI: 10. A. Wastewater Treatment. from University of Aston. pp. Girivasan. D. She has more than 5 years of Mukesh Doble is a Professor in the Department of experience in Scale up and production of protease and Laccase enzyme. P. J. Chemical engineers. Email: geegha@gmail. 1. 2.1007/s13225-011-0137. T.in. 55. “Understand the Fundamentals of Biotransformations & Bioprocesses.S. “Excellence in Basic Research” from Indian Institute of Chemical Ravishankar. Hall publ. J.. vol. five books including Green India: species composition and bioactive metabolites. Biochemical Engineering. Venkatachalam. microbes. Biomaterials. Chennai-600036.com). and M. Chemistry. Industries (ICI) and General Electric (GE) Technology centres. She has published 5 worked for 20 years in Imperial Chemical articles in international journals. K. Geetha. Wiley & sons. N. Geetha. degreesphoto in Chemical Engineering from IIT. southern or coauthored 200 technical papers. Journal of Life Sciences and Technologies Vol. U. S. 1. purification and structural elucidation of novel compounds Email: mukeshd@iitm. Springer Six-Sigma and Statistical process control. pp. Biotreatment of industrial effluents. P. His areas of interest are 1. (Tel: +91-044-2257-4107. Dye degradation. pp. from fungal extracts. U. Suryanarayanan.Production. A. Mukesh.” Botanica Marina. U. He holds B. 457-468. Biotechnology at IIT Madras.K. Doble and V. John Oct 2011.. University of Cambridge. Cyclic β-glucans from Bioremediation. New york. and filed 4 patents. Birmingham. and a member of the American and Indian Institute of 2010. Gummadi.doble0@gmail.. Bioactive compounds from INDIA. 46 . D. P. S. Prentice Diversity. Thirunavukkarasu. He is a recipient of Herdillia Award for 4. Her research interests include synthesis of mukesh.