Preliminary characterization of the thermostable dextranase producing microorganisms

Prasada Rao Vallem1 R. Nelson2, Abdallah M Jarari3Farag Elshaari3 and Jagannadha Rao Peela4
1. 2. 3. 4.

Department of Biotechnology JJ College of Arts and Sciences Pudukkottai, Tamilnadu, India, Department of Botany Govt. Arts College, Ariyalur, Tamilnadu, India, Department of Biochemistry Faculty of Medicine, Benghazi University, Benghazi, Libya. Department of Biochemistry, Faculty of Medicine, Quest International University Perak,Ipoh,Malaysia.

Corresponding Author: Dr.Prasada Rao Vallem , E-Mail:prasadvallem@yahoo.com

Abstract A Gram- positive spore forming thermophilic strict anaerobic bacterium was isolated from enrichments at 65oC with dextran as sole carbon and energy source. When compared with the total viable anaerobic micro flora, the proportion of these microbes from different areas varied over a wide range. Preliminary characterization of some of the dextranase producing microorganisms revealed a heterogeneous mixture of cell types with varying morphological and biochemical characteristics. Several bacterial isolates were identified as being members of the genus Bacillus. An additional isolate appeared to belong to the genus Bacteroides. The dextran degrading enzymes produced by these bacteria are extracellular, and a cell free preparation from one of the isolates has been shown to cause extensive endohydrolytic cleavage of high molecular weight dextrans. Key words: dextranase, dextrans, microoranism

Introduction Dextrans are high molecular weight complex polysaccharides formed, of at least 50%, by 1-6 linked glucose units, with 1-3 branch linkage and may also contain other branch linkages such as -1-2 or -1-4. The approximate average molecular weight of dextran is 5 x 106 (Inkerman et al., 1980). Dextranases catalyze the hydrolysis of the - l, 6 glucosidic bond of the polysaccharide dextran. The dextranases have often been classified as endo and exo dextranases based on the mode of action (Zevenhuizen et al., 1968). The dextran degrading enzymes have been isolated from a wide range of microorganisms with high substrate specificity. Dextranases have been obtained from a variety of sources. The dextranase producing genera include Pseudomonas, Brevebacterium, Streptococcus, Bacteroides, and Bacillus. The exoenzyme activity that releases glucose from dextran has been detected in animal tissues and bacteria (Rozenfeld et al., 1964; Henrissat, 1991; Garcia et al., 2001).

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Dextranases (1, 6--D-glucan 6glucano hydrolase) [EC 3.2.1.11] has been used in the industries in various analytical methods or measuring glucan content in sugar juices and in raw sugar (Brown and Inkerman 1992, Singlenton et al, 2001). Degradation and removal of glucans have been suggested to prevent oral diseases such as dental caries. Dextranases can inhibit the synthesis of insoluble glucans (Marotta et al., 2002). The bacteria like Bacteroides oialis (Takahashi, 1982), Flavobacterium sp. M-73 (Kobayashi et al., 1983), fungi like Pencillum funiculosum (Sugiura et al., 1973), Pencilium luteum (Fukimoto et al., 1971) and Aspergillus carneus (Hiraoka, 1972), the yeast like Lipomyces starkeyi (Koenig and Day, 1989) and Lipomyces lipofer (Ramos and Spencer-Martins, 1983) are reported to be the main producers of dextranases though the bacteria and fungi show distinct patterns of action at specific conditions like pH, Temperature, Carbon source, Protein source etc. Viewing the increasing interest in commercial production of microbial enzymes, the present study was designed on the dextranase enzyme activity pattern and to characterize the microorganisms on the basis of the production of this specific enzyme, the dextranase. Materials and Methods Samples were obtained from different places in a pre-sterilized container. Care was taken to avoid contaminating the sample. The samples were used for the detection of thermostable dextranase producing organisms. The collected samples were disrupted by using sonication for two 15-s

bursts at 30 W. Samples were quantitated turbidometrically at a wavelength of 600 nm as described previously (Gawronski. et al., 1974). Diluted sample suspensions were placed on to Tryptic soy Agar (TSA) which contained 0.5% blue dextran (Hi Media Laboratories, India), 0.5% dextran (Hi Media), 0.2% glucose, and 0.1% yeast extract. After anaerobic incubation of the plates as described above, dextranase producing microorganisms were readily identified by the presence of a decolorized zone around a colony (Fig.1). The reliability of this technique for detecting dextran hydrolysis was shown by precipitating the dextrans with absolute ethanol as described (Simonson, et al., 1972.). The areas in which dextrans failed to visibly precipitate were identical to the decolorized zone, indicating that color reduction was equivalent to dextran hydrolysis. Dextranase producing colonies were picked, streaked for isolation on the TSA medium, colony type was then restreaked on the blue dextran medium for pure culture isolation of dextranase producing microorganisms (Fig.2). The physiological parameters listed in Table 2 were determined by using the general procedures described in the Manual of Microbiological Methods (Conn, J.J. 1957) except that tryptic soy broth without glucose was used as the basal medium for the carbohydrate fermentation studies. The sulfide, indole, and motility tests were determined in SIM agar (Hi Media). Cellular morphology was determined from Gram-Stain smears of actively growing pure cultures.

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Enzymes were prepared by anaerobically growing the microorganisms in 500 ml of TS broth supplemented with 0.5% dextran and 0.1% yeast extract for 72 t0 96 h at 60oC. The cultures were chilled to 4o C and then cleared of cells by centrifugation (8,000 x g, 10 min). The cellfree supernatant was concentrated by using an ultrafiltration system with a PM10 membrane at a pressure of 50 lb/in2. The concentrated preparation was dialyzed in the cold against distilled water for 12 h, followed by dialysis against 0.01 M acetate buffer (pH 4.5). The crude enzyme preparation was clarified by centrifugation (10,000 x g, 10 min) and stored at 4 C. The dextranolytic activity of these preparations were stable for at least 2 months under these conditions. Protein was determined by the method as described (Lowry, et al., 1951). Enzyme activity was measured by monitoring the release of reducing sugar during incubation of the enzyme and substrate at 50o C. A typical reaction mixture contained 1.3 ml of dextran (20 mg/ml), and 0.2 ml of enzyme. Reducing sugar was measured (Somogyi M., 1945) on 0.1ml samples of the reaction mixtures. An alternate dextranase assay involved hydrolysis of the blue dextran incorporated into the nutrient agar medium discussed above. The assay is similar to the technique described for studies on the hydrolysis of blue starch fixed in agar by the enzyme amylase (Ceska, M. 1972., Ceska M. 1971). The size of the enzyme reaction products was determined by gel chromatography by using Bio-Gel P6 (Hi Media Laboratories, India). A 1ml sample of

a 24-h reaction mixture was placed on a column (1.5 by 25cm) which had been prewashed with 0.1M sodium acetate (pH 4.5). Carbohydrates were eluted with the same buffer, and the content of each fraction was determined by using the phenolsulphuric acid assay (Dubois, M. et al., 1956). Maltose and blue dextran at a concentration of 1mg/ml were used to calibrate the column for dead volume. Results We have taken advantage of the observation originally made by Mencier (Mencier, F. 1972) that microorganisms which produced extracellular enzyme activity capable of degrading dextrans caused a decolorization of agar media containing blue dextran. Our modification of this medium has enabled us to isolate and enumerate dextranase producing microorganisms. A typical sample suspension plated on the differential medium as shown in Fig.1. Within most of the decolorized zones one can readily determine the centrally located colony which appears to be responsible for dextranase production. In order to evaluate the iniquitousness of dextranase producing microorganisms, we obtained samples from six spots and determined the proportion of these organisms relative to the total viable anaerobic flora (Table 1). Dextranase positive organisms were present in each sample, and the quantity of these microorganisms varied between 9.16% and 3.50%. These variations do not appear to correlate with the microbial density of the individual samples. The great variation in

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colony morphology observed among the dextranase producing microorganisms indicated that a wide range of microbial types were capable of producing the enzyme. As indicated above, the microorganisms capable of producing dextranolytic enzymes were heterogeneous and varied depending upon the area from where it was obtained. To gain some insight into the type of microbes we detected by our blue dextran plating method, we selected three readily cultivable isolates. Table .2 presents a summary of some of the characteristics of these microorganisms. Isolates S1, S2 and S3 were anaerobic, irregularly shaped, and gram-positive rods. They did not exhibit detectable branching or hemolytic activity after 3 days of incubation on agar plates. The carbohydrate fermentation patterns of these organisms were similar, although none of the microorganisms could produce acid from high molecular weight dextran. In spite of the apparent similarities between these organisms, they had markedly different colonial morphologies. S1 colonies were rough, heaped-up, irregularly shaped, and white. Colonies of isolates S2 and S3 were smooth, circular, convex and white. Since the level of enzyme activity in cell free culture supernatants obtained from glucose grown cultures of several isolates was low and the residual reducing sugar content of the medium was high, we could not measure dextran hydrolysis by the reducing sugar assay. However, the blue dextran agar diffusion method was sensitive enough to allow evaluation of enzyme

activities in crude enzyme preparations. Figure 2 presents a study in which the dextranase activity in cell-free culture supernatants from isolates S1, S2, and S3 was tested along with a commercially available dextranase. It was clear that each of the preparation contained dextranolytic activity, and these results confirmed that the dextranases from the soil isolates were extracellular. We did not examined these microbes for cell bound or intracellular dextranase activity. In order to clearly demonstrate that the activities being studied actually involved in degradation of dextran, a concentrated enzyme preparation from isolate S1 was incubated with dextran, and the increase in reducing sugar was monitored. The data presented in Fig.3 illustrate that, in comparison with a heat inactivated enzyme preparation (closed circles), the S1 enzyme rapidly initiated hydrolysis of the dextran (open circles). In order to evaluate the type of mechanism by which the S1 dextranase hydrolyzed the high molecular weight dextran, we chromatographed on Bio-Gel P6 a reaction mixture similar to that obtained from the experiment in Fig.3. There was no detectable intact dextran remaining in the reaction mixture and essentially no saccharides with molecular weights less than 1,000. The majority of the carbohydrate was the form of oligosaccharides with a molecular weight ranged from 2,000 to 5,000 da.

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Discussion Use of an enriched agar medium containing blue dextran allowed us to demonstrate that a significant proportion of the anaerobic microbial flora had the capability of producing dextran degrading enzymes. The density of dextran degrading microorganisms varied from sample to sample (Table 1). We encountered two problems in our attempts to obtain an exact evaluation of the proportion of these microbes. First, although our data on the total viable anaerobic flora were comparable to the results of others (Loesche, et al., 1972), the efficiency of plating on TSA and blue dextran plates differed. Total counts on the latter were usually 20 to 50% of the TSA plates. Thus, the percentages presented in Table 1 might be low since some dextran producing microorganisms could be among those organisms that required the richer TSA medium. The second problem which we encountered in attempts to quantitate dextranase producers was that some colonies which gave clearing of blue dextran on our initial plating were refractory to subculturing. Such fastidious organisms would not grow on TSA agar or in TS broth when removed from the initial plates. Thus, we were occasionally unable to confirm that a microorganism which appeared to be a dextranase producer was really capable of yielding such activity. In spite of these difficulties, there was no doubt that the level of dextranase producing microbes varied greatly in soil from different areas. It is important to emphasize that we have looked at only a limited number of microbes in soil which appear capable of

producing dextranase. Walker (1972) has recently presented data on two streptococci from soil which produces dextran degrading enzymes. The dextranases produced by isolates S1, S2 and S3 were extracellular enzymes since these were excreted from isolated colonies of each organism and were found in cell free culture supernatants (Fig.2). Most fungal dextranases that have been investigated are extracellular (Bourne et al.,1962, Bourne, et al.,1963, Chaiet et al., 1970), although intracellular fungal dextranase has also been studied (Hutson et al., 1963), Bifidobacterium bifidus produces an extracellular dextranase (Bailey et al., 1959, Bailey et al., 1960 & 1961, Clarke 1959) and a soil bacterium has been isolated which produces at least two extracellular dextranases and some intracellular dextranase activity (Cheetham et al., 1972 Richards et al., 1972). Our preliminary studies on the action pattern of the S1 dextranase indicates that it is an endo-type enzyme resulting in the production of oligosaccharides from high molecular weight dextran. The observation that this isolate will not ferment dextran to acid suggests that little, if any, exo-type enzyme activity is produced under our growth conditions (Table 2). Interestingly, the only other bacterium which has been reported to produce an extracellular exo-type enzyme capable of degrading dextrans also belongs to the genus Bacteroides (Sery et al., 1956). Intracellular exo-type dextranase activities have been reported (Bailey et al., 1959, Zevenhuizen, L.P.T.M. 1968), and it has been suggested that the in vivo function

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of such enzymes is to degrade the oligosaccharides formed by extracellular dextranases to D-glucose (Cheetham et al., 1972). Recent studies have emphasized the role of the water insoluble, highly branched extracellular glucans from S. mutants as a major determinant in the cariogenicity of this bacterium (Guggenheim et al., 1970, & 1967). The large proportion of -(13)linkages in the S. mutants glucan makes this polysaccharide resistant to dextranases with -(16)-linkage specificity. However, Walker (Walker, G.J.1972) has presented studies which indicate a possible role for soil dextranases with -(16)-bond specificity in the regulation of water-insoluble glucan production by oral streptococci. The synthesis of streptococcal glucans containing a high proportion of -(13)linkages is sensitive to the presence of dextranases which are specific for -(16)linkages (Guggenheim et al., 1970, Walker, G.J.1972). Thus, the production of waterinsoluble soil glucans may be greatly affected by the presence of dextranases with -(16)-linkages specificity. Our findings that there are quantitative and qualitative differences in the microbes capable of producing dextranases in human decomposed soil indicate that the level of indigenous dextranase activity may play a significant role in the formation, metabolism, and pathogenicity of soil.

Acknowledgement The authors wish to express their thanks to the K.R.S. Sambasiva Rao Department of Biotechnology, Acharya Nagarjuna University, Nagarjunanagar 522510, A.P., India for providing the facilities. References 1. Bailey, R.W., and R. T. J. Clarke, 1959. A bacterial dextranase, Biochem. J. 72: 49-54. 2. Bailey, R.W., D.H. Hutson, and H. Weigel. 1960. Action of bacterial dextranase on branched dextrans. Nature (London) 186: 553-554. 3. Bailey, R.W., D.H. Hutson, and H. Weigel. 1961. The action of a Lactobacillus bifidus dextranase on a branched dextran. Biochem. J. 80:514-519. 4. Bourne, E.J., D.H. Hutson, and H. Weigel, 1962. Studies on dextran and dextranases II. The action of mould dextranases on isomaltodextrins and the effect of anomalous linkages on dextran hydrolysis. Biochem. J. 85: 158162. 5. Bourne, E.J., D.H. Hutson, and H. Weigel. 1963. Studies on dextrans and dextranases. III. Structures of oligosaccharides from Leuconostoc mesenteroids (Birmingham) dextran. Biochem. J. 86: 555-562. 6. Brown, C. F. and Inkerman, P. A. 1992. Specific method for quantitative measurement of the total dextran content of raw sugar. J. Agric. Food Chem. 40:227-233.

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7. Ceska M. 1971. Hydrolysis of a water insoluble substrate incorporated into solidified medium by enzyme -amylase contained in normal human urine, Clin. Chim Acta 33: 147-152. 8. Ceska, M. 1971. The synthesis of cross-linked dextran and its enzymatic hydrolysis. Experientia 27: 1263. 9. Ceska, M. 1972. Hydrolysis of a water insoluble substrate incorporated into solidified agar medium by enzyme -amlase contained in serum and urine of patients suffering from acute pancreatitis. Clin. Chim. Acta 36: 463-471. 10. Chaiet, L. A.J. Kemf, R. Harman, E. Kaczka, R. Wetson, K. Nollstadt, and F.J. Wolf. 1970. Isolation of a pure dextranase from Penicillium funculosum. Appl. Microbiol. 20: 421-426. 11. Cheetham, N.W.H., and G.N. Richards, 1972. Studies on dextranase. II. An intracellular bacterial dextranase. II. An intracellular bacterial dextranase. Carbohyd. Res. 25: 333-339. 12. Clarke, R.T.J. 1959. A dextranfermenting organism from the rumen closely resembling Lactobacillus Biffidus. J. Gen. Microbiol. 20:549-553 13. Conn, J.J. 1957. Manual of microbiological methods. McGraw-Hill Co. Inc., New York. 14. Critchley, P., J.M. Wood, C.A. Saxton, and S.A. Leach. 1967. The polymerization of dietary sugars by decomposed soil. Caries Res. 1:112-129.

15. Dubois, M., K.A. Gilles. J.K. Hamilton., P.A. Rebers, and F Smith. 1956. Colormetric method for the determination of sugars and related substance. Anal. Chem. 28: 350-356. 16. Fukimoto, J., Tsuji, H. and Tsuru, D. I. 1971. Penicillium luteum dextranase: its production and some enzymatic properties. J. Biochem. 69:1113-1121. 17. Garcia, B., Castellanos, A. Menendez, J. and Pons, T. 2001. Molecular cloning of an glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product. Biochem. Biophys. Res. Commun. 281:151-158. 18. Gawronski. T.H., R.H. Staat, and L.E.A. Folke. 1974. Quantitaion of human dental plaque by turbidimetry. J. Dent. Res. 52: 633. 19. Gugenheim, B., and H.E. Schroder. 1967. Biochemical and morphological aspects of extracellular polysaccharides produced by cariogenic streptococci. Helv. Odontol. Acta. 11: 131-152. 20. Guggenheim, B. 1970. Enzymatic hydrolysis and structure of water insoluble glucan produced by glucosyltransferases from a strain of Streptococcus mutans. Helv. Odontol. Acta 5(Suppl. 14) 89108. 21. Henrissat, B. 1991. Aclassification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280: 309-316

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22. Hiraoka, N., J. Fukumoto, and D. Tsurru. 1972. Studies on mold dextranases. III. Purification and some enzymatic properties of Asperigillus carneus dextranase J. Biochem. 71: 57-64. 23. Hutson, D.H., and H. Weigel. 1963. Studies on dextrans and dextranases. IV. Mechanisms of the actions of intra-and extra cellular mould dextranases. Biochem. J. 88: 588-591. 24. Inkerman, P.A. 1980. An appraisal of the use of dextranase. In proceedings of the international Society of sugar cane Technologist 17th Congress. Pp.2411-2427. Manila, Philippines. 25. Kobayashi, M., Tagaki, S. Shiota, M. Mitsuishi, Y. and Matsuda, K. 1983. An isomaltotriose-producing dextranase from Flavobacterium sp. M-73: Purification and properties. Agric. Biol. Chem. 47:2585-2593. 26. Koenig, D. W. and Day, D. F.1989. Induction of Lipomyces starkeyi dextranase. Appl. Environ. Microbiol. 55:20792081. 27. Loesche, W.J., and R.J. Gibbons, 1965. A practical scheme for identification of the most numerous oral gram negative anaerobic rods. Arch. Oral Biol. 10: 723-725. 28. Lowry. O.H., N.J. Rosebrough, A.L. Farr,a nd R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

29. Marotta, M., Martino, A., Rosa, A., Farina, E., Carteni, M. and Rosa. M. 2002. Degradation of dental plaque glucans and prevention of glucan formation using commercial enzymes. Process Biochem. 38:101-108. 30. Mencier, F. 1972. Methods simple et rapide de mise enevidence des microorganisms producteurs de dextranase. Ann. Inst. Pasteur 122. 153-157. 31. Ramos, A. and Spencer-Martins, I. 1983. Extracellular glucoseproducing exodextranase of the Lipomyces lipofer. Antonie van Leeuwenhoek 49:183-190. 32. Richards, G.N., and M. Streamer. 1972. Studies on dextranases. I. Isolation of extracellular, bacterial dextranases. Carbohyd. Res. 25: 323-332. 33. Rozenfel'd, E. L. and Saenko, A. S. 1964. Metabolism in vivo of clinical dextran. Clin. Chim. Acta 10:223-228. 34. Sery, T.W., and E. J. Hehre, 1956, Degradation of dextrans by enzymes of intestina bacteria. J. Bacteriol. 71: 373-380. 35. Simonson, L.G., B.L. Lamberts and I.L. Shklair. 1972. A rapid plate method for screening dextranase producing microorganisms. J. dent. Res. 51: 675. 36. Singlenton, V., Horn, J., Bucke, C. and Adlard, M. 2001. A new polarimetric method for the analysis of dextran and sucrose. Int. Sugar J. 103:251-254. 37. Somogyi. M. 1945. A new reagent for the determination of sugars. J. Biol. Chem 160: 61-68.

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38. Sugiura, M., Ito, A., Ogiso, T., Kato, K. and Asano, H. 1973. Purification of dextranase from Penicillium funiculosum and its enzymatic properties. Biochim. Biophis. Acta 309:357-362. 39. Takahashi, N. 1982. Isolation and properties of dextranase from Bacteroides oralis Ig4a. Microbiol. Immunol. 26:375-386.

40. Walker, G.J. 1972 Some properties of dextranglucosidase isolated from streptococci and its use in studies on dextran systhesis. J. Dent. Res. 51: 409414. 41. Zevenhuizen, L.P.T.M. 1968. Cell-bound exo-dextranase of Bacillus species, Carbohyd. Res. 25: 310-318.

Fig.1. Visualization of dextranase producing colonies on enriched nutrient agar medium.

Fig.2. Hydrolysis of blue dextran by dextranases. Isolate was streaked and the plates were incubated at 50o C for 24 h

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Fig 3. Release of reducing sugar from dextran by an enzyme preparation from isolate S1

Table.1. Enumeration of dextranase producing microorganisms Viable microorganisms/mg of soil (wet wt) 5.8 X 108 1.2 x 108 4.3 x 107 5.0 X 108 8.5 x 107 5.7 x 107 Dextranaseproducing colonies (%) 0.45 0.29 0.16 0.32 0.78 3.5

Sample

1 2 3 4 5 6

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Table.2. Characteristics of some dextranase producing microorganisms

Characteristic Cellular morphology Gram reaction Oxygen relationship Motility Catalase test Indole production Nitrate reduction Gelatin Liquefication H2S production Red blood cell hemolysis a Carbohydrate fermentation: b Glucose Mannose Maltose Starch Dextran Esculin Lactose .
a b

S1 Irregular rods + Anaerobe + + + +

Isolate S2 Irregular rods + Anaerobe + + +

S3 Irregular rods + Anaerobe + + +

denotes weak hemolysis after prolonged incubation (6days). + denotes pH 4.0 to 5.0; denotes pH 5.0 to 6.0 and denoted pH > 6.0.

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