ASSAY for TRANSGLYCOSYLATION REACTION of Xanthomonas Campestris on CARBOHYDRATE SOURCES

ASSAY FOR TRANSGLYCOSYLATION REACTION OF Xanthomonas campestris ON CARBOHYDRATE SOURCES

JOKO SULISTYO1, RINI HANDAYANI2, RITA DWI RAHAYU2

1Faculty of Food Science and Nutrition, University Malaysia Sabah, Malaysia 2Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences, Indonesia Email: [email protected] and [email protected]

ABSTRACT

Phytopathogenic bacterial Xanthomonas campestris causes rot disease in cruciferous plants, such as peppers, tomatoes, broccoli, and cabbage. On agar medium, this bacterial culture is able to produce extracellular biopolymer that can be utilized as an ingredient in food preparation that is known as xanthan gum. This polysaccharide can be applied in commercial industry as thickening agents, viscosifier, stabilizer, emulsifier and suspension agent. This study aims to determine ability of X. campestris culture in synthesizing such kind of a simple polysaccharide as a transfer product of transglycosylation reaction using extracelluler enzyme of this bacterial culture on several sources of carbohydrates, in order to study process of enzymatic synthesis of this xanthan gum. Transfer products were tested using thin layer chromatography and high performance liquid chromatography. The results showed that corn flour was the best substrate on formation of the transfer product compared to other sources of carbohydrate as its substrate. The result showed that corn flour could use xylose as its acceptor to produce the transfer product as amount of 0.47% compared to standard of xanthan gum.

Key words: gum xanthan, enzymatic synthesis, Xanthomonas campestris, transglycosylation, transfer product.

1. INTRODUCTION polysaccharide known as xanthan gum that can be used for food processing industry. Industrial Bacterium Xanthomonas campestris is a member application of xanthan gum is as a stabilizer, of family aerobic bacteria Xanthomonadaceae viscosifier, thickener, and suspension agent which causes rot disease in cruciferous plants, such (Kennedy and Bradshaw, 1984, Rosalam and as peppers, broccoli, and cabbage (Onsando, 1992). England, 2006).

Characteristics of X. campestris is to have one Xanthan gum is a kind of water-soluble hetero - - flagella, can not reduce NO3 into NO2 , can not polysaccharide produced by these Gram-negative play a role in decarboxylation of lysine, methionine bacteria (Borges et al, 2009). It is a kind of or cysteine which is required for its growth. biopolymer that has bright prospects since it has a Mucoid is formed on nutrient agar medium high commercial value for some industrial containing 5% glucose. It is able to hydrolyze application purposes. It is applicable as suspending gelatin and starch, and hydrolyzes esculine and agent to remove pulp and materials that can make proteins to produce H2S from peptone, and shows murky of some beverages products and also is used pectinase activity during fermentation. The as stabilizer for oil emulsions of certain beverage maximum temperature to grow is 35-39ºC, and products, frozen foods, and in non-food industry, shows tolerance to salinity, producing acid as well such as textile industries and oil welldrilling fluid as pathogenicity against crops, especially group of additive of mining industries. A commercial price cruciferous (John et al, 1994). This bacterium was of xanthan gum is very expensive, since it is able to produce extracellular enzymes such as produced by using basic ingredients of glucose and amylase, endoglucanase, polygalacturonate lyase sucrose (Yoo and Harcum, 1999). The annual and protease (Dow and Daniels, 1994). world production of xanthan gum is estimated at approximately 25.000 tonnes (Galindo, 1994). X. campestris is also able to produce extracellular

2. MATERIALS AND METHODS Xanthan Gum is tangible granules when dry and is able to increase the viscosity of water with a 2.1. Preparation of Media for X. campestris concentration of only 0.01%. It has a molecular weight that varies between 2000-62000 kDa. Culture of X. campestris was initially grown in Width of this range due to influence of hydrogen steril Lauria medium containing 5g yeast extract, bonds that stabilize polymer aggregates in water. 10 g tryptone, 5g NaCl, and 15g agar. Furthermore, One monomer of xanthan gum containing five culture of X. campestris was transferred into units consisting of two units of glucose, two units medium containing 3g (NH)2SO4, 0.5g MgSO4, of mannose, and one unit of glucuronic acid 0.1g CaSO4, 0.01g MnSO4, 0.1g FeSO4, 3g malt (Kennedy and Bradshaw, 1984). extract, 0.5g peptone, and 1% glucose as initial substrate in 0.05 mM Na-phosphate buffer at pH 7, The mechanism on formation of xanthan gum is and incubated at 37oC for 4-5 days. Furthermore, very complicated and does not well known. culture of X. campestris was divided and then However, therefore synthesis process of xanthan poured into several Erlenmeyer flasks (100 ml) gum can be simply examined through enzymatic containing some kind of carbohydrate as substrates transglycosylation reactions. (2%) were cassava, potatoes, sweet potatoes, maize and rice flour, respectively, that had been sterilized Enzyme cyclodextrin glucanotransferase (1,4-α-D- at 121oC for 20 min in advanced. 1.4-glukano-transferase, EC 2.4.1.19) abbreviated as CGTase is enzyme that catalyzes the synthesis 2.2. CGTase Enzyme Production of starch through cyclization reaction. This enzyme belongs to transferase enzyme that plays a role in Media for the production of enzyme CGTase that removal of glycosidic groups in the cyclization has been inoculated with culture of X. campestris reaction of dextrin into cyclodextrins (Kometani et was incubated for 72-120 h, using shaker incubator al, 1994; Mori et al, 1994). at 240 rpm and 37°C, then centrifuged at 8030g for 15 min at 4°C. Furthermore, supernatant was used CGTase is a multifunctional enzyme that plays a as a source of crude CGTase (Sulistyo et al, 2002). role in converting starch and α-1,4 glucan into cyclodextrin via intramolecular transglycosylation 2.3. Enzymatic Activity Test for CGTase reaction. This enzyme is also able to catalyze coupling reaction that opening cyclodextrin ring Determination of optimum conditions of CGTase and transferring linear maltooligosaccharides was carried out at various pH and temperature. The through via intermolecular transglycosylation reaction mixture consisting of 100 mL crude reaction to a appropriate acceptor. CGTase solution and 450 mL of 50 mM Na- phosphate buffer containing 0.5% soluble starch CGTase is not only catalyzes converting starch was incubated at 45°C for 10 min. The reaction into cyclodextrins intramolecularly and transfer was stopped by addition of 1 ml of HCl 0,5N. The glucosyl group intermolecularly to the appropriate reaction mixture was then treated with 2.5 ml of acceptor, however it can also hydrolyzes starch and iodine dye (0.05% KI containing 0.005% I2). cyclodextrin into simpler compounds (Kometani et Absorbance was measured using a spectro- al, 1996). CGTase is produced by Bacillus photometer at 660 nm. One unit of enzyme activity macerans, B. megaterium, B. circulans, B. cereus is expressed as amount of enzyme that may (Tankova, 1998), and there is also possibility could degrade as much as 0.5 unit of absorbance at 660 be extracted from culture of X. campestris. nm (Sulistyo et al, 2002). We then performed a series of measurement for five days on every 24 h. Therefore in this study X. campestris was grown on a culture media to produce CGTase that has 2.4. Assay for Density of Carbohydrate capacity regarding with transglycosylation reaction in order to synthesize transfer product which has Test of carbohydrate content was determined using similarity to the xanthan gum. phenol-sulfuric acid method (Dubois et al, 1956). A total of 0.5 ml of 5% phenol plus 2.5 ml of

concentrated H2SO4, and added with 0.5 ml of samples that have been diluted 10 times with distilled water. The reaction mixture was left for 20

Oct. 2014. Vol. 2, No.6 ISSN 2311 -2476 International Journal of Research In Agriculture and Food Sciences © 2013 - 2014 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html min at room temperature, then absorbance value buffer at pH range between 6.0 to 8.0. The results was determined at 490 nm. Carbohydrate content showed that the highest CGTase activity was at was determined using a standard curve of soluble pH7 which was equal to 0.4522 U/ml (Fig. 1). starch. The standard curve was prepared by measuring the absorbance of carbohydrate content Determination of optimum temperature of enzyme where the concentration was predetermined. activity of CGTase derived from X. campestris was Concentration of standard solution was prepared by carried out at a temperature of pH 7.0 in 0.5 mM dissolving soluble starch at 0, 10, 20, 30, 40, and phosphate buffer at temperature range between 50 mg, in distilled water, respectively, until the 35oC to 70 oC. The results showed that the highest volume of the solution up to 5 ml. The solution is CGTase activity was at 45oC which was equal to then reacted with 0.5 ml of phenol and 2.5 ml of 0.3472 U/ml. Enzyme activity was decreased at 50 concentrated H2SO4. Subsequently the absorbance to 55°C, but showed a slight increased at 65°C that of reaction mixture was measured at 490 nm, was equal to 0.2658 units/ml. (Fig. 2). ranging from smallest to highest concentration. 0.50 2.5. Assay for Activity of Transglycosylation 0.40 The reaction mixture (2 ml) consist of 1.5 ml of 0.30 0.05M Na-phosphate buffer pH 7, containing 5% 0.20 soluble starch and 2% resorcinol and 0.5 ml of crude CGTase solution was incubated at 45ºC for 0.10 24 h. Activity of enzyme was stopped at 100°C for Enzyme Activity (U/ml) Activity Enzyme 0.00 10min and cooling down, and added with enzyme 6 6.5 7 7.5 8 of α-amyloglucosidase as much as 0.005%, and incubated again for 45 min. The resulting of Influence of pH reaction products were identified by thin layer Figure 1. Enzymatic activity of CGTase derived from X. chromatography (TLC) developed by using a campestris at various pH range. solution of ethyl acetate-acetic acid-distilled water (3:1:1, v/v). TLC plate was then burned at 100°C 0.40 for 1 h, and then sprayed with a reagent containing 20% H2SO4 in methanol, and then burned again at 0.30 150ºC for 5-10 min. Having obtained a substrate with the highest transfer reaction activity, then 0.20 transfer activity was tested again by replacing the resolsinol as acceptor with a simple sugar such as 0.10 glucose, galactose, xylose, and fructose untill the highest of transfer products could be obtained. Activity (U/ml Enzyme 0.00 35 40 45 50 55 60 65 70 2.6. Analysis for Xanthan Gum Influence of Temperature ( C)

Reaction mixture consisting of corn flour as Figure 2. Enzymatic activity of CGTase derived from X. substrate and xylose as acceptor was incubated campestris at various temperature range. with CGTase and the reaction product was then analyzed using HPLC with carbohydrate column Determination of optimal conditions for hydrolytic PLH-2 with the solvent of acetonitrile : water activity of CGTase, including the determination of (80:20, v/v) at 40ºC. Standard solution used was a the optimally pH and temperature condition which commercial xanthan gum which was dissolved at a is required by the enzyme to be active. The highest concentration of 0.6%. hydrolytic activity at a certain temperature and pH shows the optimum activity of the enzyme. 3. RESULTS AND DISCUSSION The Enzyme CGTase derived from X. campestris

was incubated at various temperatures ranging 3.1. Influence of pH and Temperature Against from 35°C to 70°C, and the highest activity was Enzyme Activity of CGTase shown at 45°C as amount of 0.3472 units/ml. While for the determination of the optimum pH of Determination of optimum pH of enzyme activity enzyme showed that the highest CGTase activity of CGTase derived from X. campestris was carried was at pH 7 as amount of 0.4522 units/ml. The out at a temperature of 45ºC in 0.5 mM phosphate 3

Oct. 2014. Vol. 2, No.6 ISSN 2311 -2476 International Journal of Research In Agriculture and Food Sciences © 2013 - 2014 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html results seemed to be in accordance with what had concentration of media containing rice starch been reported by Mori et al (1994), that the activity decreased to 236.65 g/ml (Figure 4). of CGTase was stable at ≤50°C and at pH 6-10. Assay for the hydrolytic activity of CGTase was Buffer that had been used for production of the performed by using starch as its substrate. The CGTase was phosphate buffer. Phosphate is an ability of enzyme to hydrolyze starch was tested by inorganic component which can be used for adding 0.05% KI solution containing 0.005% I2, buffering medium in the range of normal pH that is and therefore when the reagent was reacted with important to maintain a physiological balance of starch, it formed a blue color. The intensity of blue microbes. In addition, phosphate is also as source color which was formed is related to amount of of phosphorus that is not harmful to microbes. starch which is not hydrolyzed, and therefore that more starch is hydrolyzed, then the intensity of the blue color of solution will be fading. 3.2. Influence of Substrates Against Enzyme Activity of CGTase 0.4 The measurement of CGTase activity showed that 0.4 there has been varies in increasing of activity. It 0.3 was increased in the present of potato, corn and 0.3 cassava starch as its substrates on the first 24 h and tend to stable up to 120 h of incubation. However, 0.2 the enzyme activity was increased gradually on 0.2 24h to 120 h in the present of substrate of rice and sweet potato (Fig. 3). 0.1 Cassava Potato Sweet Potato Corn 0.1 Rice Cassava 1.6 (mg/ml) Substrate of Hydrolysis Potato 0.0 1.4 Sweet Potato Corn 0-h 24-h 48-h 72-h 96-h 120-h 1.2 Rice Incubation Time

ase (U/ml) ase 1.0 Fig. 4. The performance of enzyme to hydrolyze carbon - sources on production medium containing several kinds 0.8 of commercial starchs. 0.6 0.4 3.3. Activity of Transglycosylation Reaction

Activity of CGTActivity 0.2 0.0 Assay for transglycosylation activity was carried 0-h 24-h 48-h 72-h 96-h 120-h out on reaction mixture containing various carbon Incubation Time sources as its substrates, which could be transferring glycosyl groups to polyphenol as Figure 3. Enzymatic activity of X. campestris CGTase acceptor. The enzymatic reactions of each reaction towards several kinds of carbon sources. mixture were analyzed using TLC. The existence of a transfer product is determined as a Testing ability of enzyme in hydrolysis substrate of chromatogram spot having Rf spot is parallel with carbohydrate was carried out by performing the Rf spot of arbutin, which is used as a marker spot. enzyme in media containing substrate of cassava The result showed that the enzymatic reaction potatoes, sweet potatoes, corn, and rice flours, using corn starch as its substrate obtaining a respectively, for 5 days and measured at every 24h thickest spot compared to other similar spots of of incubation. The result showed that concentration reactions mixtures containing other starchs as their of each tested substrates have decreased. The substrate (Fig. 5). concentration of media containing cassava starch decreased as amount of 168.78 g/ml, while the Enzymes that could be extracted was a crude concentration of media containing potato starch extract of CGTase, although allegedly contained decreased to 174.04 g/ml. The concentration of some other enzymes those could be also produced media containing sweet potato decreased to 153.20 by using starch as their inducer, such as α-amylase, g/ml, and the concentration of media containing cellulose, glucosidase, etc. However, both of corn starch decreased to 100.05 g/ml, while the enzymes, α-amylase and CGTase could be furthermore distinguished by carrying out testing

Oct. 2014. Vol. 2, No.6 ISSN 2311 -2476 International Journal of Research In Agriculture and Food Sciences © 2013 - 2014 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html for their capacity on transglycosylation reaction activity, since the α-amylase catalyzes only Fig 5. Thin layer chromatogram of reaction mixture hydrolytic activity, whereas the CGTase catalyzes products containing various carbon sources as substrate hydrolytic activity and also transglycosylation and polyphenol (resorcinol) as acceptor. (CaF, cassava reaction activity at once (Kometani et al. 1996). flour; PoF, potato flour; SPF, sweet potato flour; CnF, corn flour; RcF, rice flour).

3.4. Assay for Transfer Products Similar to The product of transglycosylation reaction in Xanthan gum addition to generate a transfer product in structure of polyphenol glucoside, there is also a possibility This bacterial CGTase was produced in medium in the structure of polyphenols-glucobioside or containing 2% starchs as its substrate. The starchs polyphenol-glucotrioside as its side products of those had been applied also as its inducer for reaction (Soeka and Sulistyo, 2007). However, producing such of enzyme which has capacity to both of these side products can be catalyzed into play a role on transglycosylation reaction were polyphenol glucoside by addition of enzyme cassava, potato, sweet potato corn, and rice. amyloglucosidase which is able to hydrolyze these Assay for transglycosylation reaction activity of two compound of side products from non-reducing the enzyme CGTase derived from some culture end sequentially and consequently releasing units media containing some kinds of carbohydrates as of glucose which is bond to other glucosyl group. carbon source corresponding to enable transferring glycosyl to acceptor polyphenol on synthesis of Culture of X. campestris exhibited capacity for polyphenol-glycosides as transfer product which transfer reaction as shown by the presence of spot was determined by using TLC and identified by on TLC derived from its culture media which presence of spot which showed of Retention factor showed the Rf value closed to Rf value of arbutin at (Rf) value was approaching to Rf value of arbutin. Rf 0.78. However, further observation showed that one out of five media containing carbohydrates Arbutin is a polyphenol glycoside compound is those were tested, that was only media containing known as 4-hydroxyphenyl-D-glucanopyranoside, corn starch as its substrate which showed the and has been reported to have biological activities thickest spot of the transfer product. Subsequently, as antimicrobial, antiinflamantori, and antioxidant the culture medium containing the corn starch was compound (Handayani and Sulistyo, 2008). furthermore used as the selected media for producing such the transfer product by replacing Stationary phase used in TLC plates was silica the acceptor of polyphenol with some simple which is polar material, whereas for mobile phase sugars including glucose, fructose, xylose, and is developing solution which consisted of ethyl galactose as its acceptors. acetate, acetic acid, and distilled water (3:1:1, v/v). A compound with a lower polarity will be carried Assay for the ability of enzyme to catalyzes the along with the mobile phase so it is detected to synthesis of transfer product by using some kind of have a high Rf value, whereas compound with a monosaccharides as acceptors (glucose, fructose, higher polarity will be retained, and therefore it xylose and galactose), resulted in the formation of tends to have lower Rf value. two spots reaction producs that were detected on TLC plate which was derived from the reaction mixture containing xylose as acceptor. Of the four sugar-acceptor was used, the result showed that the reaction, the transfer of products from the reaction mixture containing xylose acceptor, indicating the spot chromatogram, the Rf of the spot closest to the spot of arbutin as a marker (Fig. 6).

Based on the measurements of Rf values of thin layer chromatogram, it could be presumed that one of the two spots was a spot of hydrolysis product (Rf 0.45), which was parallel to the Rf of glucose which was used as standard solution, while another spot which was corresponding to Rf spot of methyl- α-glucoside (Rf 0.58) as standard solution.

Concentration of the transfer product similar The results indicated that there was kind of xanthan gum which was contained in the sample glycoside as transfer product which had a bond solution could be calculated by ratio of the peak containing glucosyl and xylosyl group. It has not area of the standard solution containing xanthan been clearly revealed whether the transfer product gum compared to the peak area of the sample is similar to xanthan gum or other transfer product. solution, and multiplied by the concentration of the Whereas the reaction mixture containing the other standard solution. The result showed that the acceptors could only be able to produce single spot sample solution which was contained the transfer those were spot of glucose (Rf 0.43), fructose (Rf product was similar to the Rt of xanthan gum. 0.45), and galactose (Rf 0.45), respectively. Analysis of carbohydrate content produces a reaction among of carbohydrate with phenol and concentrated sulfuric acid, so it will form a dark color when the carbohydrate content is very high, while the lower of carbohydrate content leads the color of reaction mixture is more faded.

Peak of Xanthan Gum (Rt : 1.012 min)

Peak of Transfer Product (Rt : 0.998 min)

Fig 6. Thin layer chromatogram of reaction mixture products containing various carbon sources as acceptor Fig 7. Chromatogram of HPLC for standard solution (Glucose, Fructose, Xylose, Galactose). containing xanthan gum compared to sample solution containing transfer product similar to xanthan gum. Determination for transfer products of enzymatic reactions was carried out using CGTase that had Analysis on carbohydrate content of culture media been applied into production medium containing was conducted over five days at every 24h. We corn starch as the substrate and sugar xylose as the have observed that all of respective culture media acceptor. To detect any transfer products was were found to be decreased in the concentration of formed, a commercial xanthan gum (0.6%) was carbohydrates. It was occurred due to character of used as a standard solution. The result of analysis X. campestris as a kind of chemoorganotropic using HPLC showed that the standard solution of microorganism which is capable of using a wide xanthan gum has a peak of chromatogram at a variety of carbohydrates and organic acids salts as retention time (Rt) 1.012 min, while the retention a source of carbon (John et al., 1994). time of sample solution was 0.998 min (Fig. 7). Analysis of the reaction products by using HPLC Result of analysis by using HPLC indicating that on samples derived from the reaction mixture the content of transfer product that is similar to containing corn starch as substrate and xylose as xanthan gum which was detected from the reaction acceptor was needed to be done giving the analysis mixture by application of this reaction was 0.47%. by TLC has not been satisfied to reveale regarding Kerdsup et al (2007) reported that production of with presence or absence of the transfer products xanthan gum by using a wild type culture of X. which was similar to xanthan gum could be campestris and cassava flour as its substrate could synthesized by transglycosylation reaction or not. yield 4.31 g/L of xanthan gum and the yield could However, analysis of structural similarity of the still be improved to reach 5.97 g/L. transfer products with more complex structure of

Oct. 2014. Vol. 2, No.6 ISSN 2311 -2476 International Journal of Research In Agriculture and Food Sciences © 2013 - 2014 IJRAFS & K.A.J. All rights reserved http://www.ijsk.org/ijrafs.html xanthan gum requiring identification procedure by cyclodextrin glucanotransferase from an using more specific laboratory equipment. alcalophilic Bacillus species in alcaline pH and propeties of hesperidin glycosides. J Biosci Biotech Biochem 58: 1990-1994. 10. Kometani, T., Y. Terada, T. Nishimura, T. ACKNOWLEDGMENTS Nakae, H. Takii and S. Okada (1996). Acceptor specificity of cyclodextrin We are thankful to Mr. Dedy Sanji Prastiko, Astra glucanotransferase from an alcalophilic Agro Lestari Co.Ltd. for the provision of research Bacillus species and synthesis of glucosyl data and Microbiology Division, Research Center rhamnose. J Biosci Biotech Biochem 60: for Biology, Indonesian Institute of Sciences for 1176-1178. the laboratory facilities. 11. Mori, S., S Hirose, T. Oya and S. Kitahana (1994). Purification and properties of REFERENCES cyclodextrin glucanotransferase from Brevibacterium sp. No. 9605. J Biosci 1. Borges, C.D., R.C.M. Paula, J.P.A. Feitosa, Biotech Biochem 58: 1968-1972. and C.T. Vendruscolo (2009). The influence 12. Onsando, J.M. (1992). Black rot of crucifers. of thermal treatment and operational Di dalam: Chaube H S, Kumar J, conditions on xanthan produced by X. Mukhopadhyay AN, Singh US, and Cliffs E, arboricola pv pruni strain 106. Carbo Pol. editor. Plant Diseases of International 75: 262–268. Importance II: Diseases of Vegetable and Oil 2. Dow, J.M. and M.J. Daniels (1994). Seed Crops. New York: Prentice Hall. hlm Pathogenicity determinants and global 243–252. regulation of pathogenicity in Xanthomonas 13. Rosalam, S, and R. England (2006). Review campestris pv. campestris. In : Dangl J.L, of xanthan gum production from unmodified editor. Molecular and Cellular Mechanisms starches by Xanthomonas campestris sp. J in Bacterial Pathogenesis of Plants and Enzyme Microb Technol 39: 197-207. Animals. Berlin: Springer. pp 29–41. 14. Sulistyo, J., R. Handayani and M. Hawab 3. Dubois, M., L.A Gilles, J.K. Hamilton, P.A. (2002). Antioxidant activity of polyphenol Robers and F. Smith (1956). Colormetric glycosides synthesized by transglycosylation method for determination of sugar and related reaction of Bacillus macerans CGTase. substances. Anal Chem 28: 350-356. BioSMART 4: 18-22. 4. Galindo, E. (1994). Xanthan gum World 15. Soeka, Y.S. and J. Sulistyo (2007). Market Report. Mexico City: Institute de Antimicrobial activity of flavonoid- Biotechnology, Universided Nacional glycosides synthesized by enzymatic Autonomo De Mexico. transglycosilation reaction. Berita Biologi. 8 5. Handayani, R. and J. Sulistyo (2008). (6) : 455-464. Synthesis of flavonoid -glycoside through 16. Tankova, A. 1998. Bacterial cyclodextrin enzymatic transglycosylation reaction and glucanotransferase. J Enzyme Microb Technol assay of its antioxidant activity. Biodiversitas. 22: 678-686. 9 (1): 1- 4. 17. Yoo, S.D. and S.W. Harcum (1999). Xanthan 6. John, G.H., R.K, Noel, H.A.S. Peter, T.S. gum production from waste sugar beet pulp. James, T.S. Williams (1994). Determinative Biores Technol 70: 105–109. Bacteriology. Ed 9th. Baltimore: Williams and Wilkins. 7. Kennedy, J.F., and J.I. Bradshaw (1984). Production, properties and applications of xanthan. Proc Indust Microb 19: 319-371. 8. Kerdsup, P., S. Tantratian, R. Sanguandeekul, and C. Imjongjirak (2009). Xanthan production by mutant strain of Xanthomonas campestris TISTR 840 in raw cassava starch medium. Food Bioprocess Technol. 250: 7-9. 9. Kometani, T., Y. Terada, T. Nishimura, T. Nakae, H. Takii and S. Okada (1994). Transglycosylation to hesperidin by 7