SOME ASPECTS OF CARBOHYDRATE METABOLISM AND PRODUCTION OF GLYCOSYLTRANSFERASES FROM MUTANT STRAIN LEUCONOSTOC MESENTEROIDES M2860

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1 SOME ASPECTS OF CARBOHYDRATE METABOLISM AND PRODUCTION OF GLYCOSYLTRANSFERASES FROM MUTANT STRAIN LEUCONOSTOC MESENTEROIDES M2860 T. Vasileva 1, V. Bivolarski 1, I. Ivanova 2 and I. Iliev 1 1 Plovdiv University, Department of Biochemistry and Microbiology, Plovdiv, Bulgaria 2 Sofia University, Department of General and Applied Microbiology, Sofia, Bulgaria Correspondence to: Tonka Vasileva tonika1@abv.bg ABSTRACT The production of glycosyltransferases (GTFs) from constitutive mutant strain Leuconostoc mesenteroides M2860 was studied. When grown in glucose medium in the absence of sucrose Leuc. mesenteroides M2860 produced low, but detectable GTF activity. Much of the GTF activity (81%) in sucrose grown cultures was located in the cell pellets. Extracellular and cell associated activities were determined when run on SDS-PAGE for in situ activity detection by periodic acid-schiff s staining. In situ analysis showed single band corresponding to 180 kda molecular size in supernatant and enzyme concentrate, received by cultivation on glucose media. The enzyme concentrate and supernatant fraction obtained by fermentation on sucrose media showed three bands corresponding to 180 kda, 120 kda and 86 kda. Keywords: constitutive mutant, glycosyltransferases, Leuconostoc mesenteroides Introduction Lactic acid bacteria Leuconostoc mesenteroides produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs). GTFs synthesize glucans from sucrose by transferring glucosyl units to nascent glucan chains and liberating fructose. FTFs synthesize levans from sucrose, producing glucose as a byproduct. The GTFs of Streptococcus sp. are produced constitutively, but those from Leuc. mesenteroides are inducible enzymes requiring sucrose for induction (16, 13). Neely and Nott, Kim and Robyt did not detect GTF activity in supernatant fractions of cultures grown in media with sugars other than sucrose (13, 9). The presence of sucrose in cultural media causes problems because the enzyme remains in an aggregated form in the presence of glucan resulting in a high molecular weight. The presence of glucan in the culture media causes some problems because of its increased viscosity, and the enzyme is difficult to purify (10). Most of difficulties for purification of GTFs from Leuc. 571 mesenteroides are overcome by the use of constitutive mutants that can produce GTFs in the absence of sucrose. Kitaoka and Robyt have been selected several high producing constitutive mutants for GTFs from Leuc. mesenteroides B-512 FMC, B-742 CB, B-1299 CB and B C (10). Smith and Zahnly have been described also mutants of Leuc. mesenteroides B-1355 constitutive for synthesis of GTFs, after growth in glucose media (18). The isolation of the constitutive mutants of Leuc. mesenteroides strains provides a means of purifying their GTFs in the absence of glucan, especially for those strains that elaborate GTFs producing glucans resistant to endodextranase treatment (9). The production of these enzymes in the absence of glucan also can greatly facilitate the study of the properties and mechanism of action of the GTFs. The aim of the present work is to study the production of GTFs from constitutive mutant strain Leuc. mesenteroides M2860 when grown on media containing glucose and sucrose and to identify the produced extracellular and cell associated enzymes.

2 Materials and Methods Bacterial strains and culture media Leuconostoc mesenteroides M2860 is a mutant, obtained by chemical mutagenesis of strain 28. The strains were cultured 6-8h in GTF culture media at 27 C on a rotary shaker (200rpm) (7). Biomass measurements Bacterial growth was measured by a turbidimetric method at 620 nm and calibrated against cell dry-weight measurements as previously described (8). Purification of glucosyltransferase GTF was separated from the supernatants and concentrated by using aqueous two-phase partition between dextran (native or exogenous) and polyethyleneglycol (PEG) (14). After addition of PEG-1500 to get the final concentration of 20% (w/v), the dextran rich phase containing GTF was separated by centrifugation at 7000 x g for 20 min at 4 C, collected in the pellet, and diluted in 20 mm sodium acetate buffer, ph 5.4. Glucosyltransferase assay One unit of glucosyltransferase is defined as the amount of enzyme that catalyzes the production of 1 mol of fructose per min. at 30 C in 20 mm sodium acetate buffer, ph 5.4, with 100 g of sucrose per liter, 0.05 g of CaCl 2 per liter, and 1 g of NaN 3 per liter. It was ascertained that the reducing sugar measured by DNS assay was due to glucosyltransferase and not to levansucrase, inveratase, or sucrose phosphorylase activity as described by Dols, et al. (3, 4). Glucose concentration was measured enzymatically by glucose oxidase, using a Beckman Glucose Analyzer 2. Protein determination Proteins were assayed by method of Bradford (1). Electrophoresis analysis SDS-PAGE (70 x 80 mm slab gels, 7 % acrilamide gels) was conducted by the method of Laemmli (11). The proteins were stained with Coomassie Brilliant Blue R 250 (Sigma Chemical Co.). Glucosyltransferase activities were detected by incubating the gels in 10% sucrose overnight, followed by staining for polysaccharide by a periodic acid-schiff s procedure (12). Myosin, α-macroglobulin, β-galactosidase and transferin were used as molecular mass protein standards. Analysis of metabolites Lactic acid was determined enzymatically with L-lactate dehydrogenase and D-lactate dehidrogenase (commercial available kit code , Boehringer, Mannheim, Germany). Acetic acid was determined enzymatically with acetyl-coa synthetase, citrate synthase and malate dehydrogenase (commercial available kit code Boehringer, Mannheim, Germany). Ethanol was determined enzymatically with alcohol dehydrogenase and aldehyde dehydrogenase (commercial available kit code Boehringer, Mannheim, Germany). Results and Discussion In our previous study, we have isolated a constitutive mutant strain Leuc. mesenteroides M2860 of a parent strain Leuc. mesenteroides 28. We have shown that the mutant produces detectable extracellular GTF activity with a molecular mass of 180 kda when glucose replaces sucrose as a growth substrate (7). In the present work, we compared fermentation profiles of GTFs produced by mutant strain M2860 in media containing glucose and sucrose as a carbon sources. On the Fig. 1 and the Fig. 2 are presented the results of fermentation profiles for the growth and GTF production of Leuc. mesenteroides M2860 on media containing sucrose and glucose, respectively. Fig. 1. Glucosyltransferase activity of mutant strain Leuc. mesenteroides M2860, cultivated in glucose media Fig. 2. Glucosyltransferase activity of mutant strain Leuc. mesenteroides M2860, cultivated in sucrose media The maximum of activity was determined at the 5.5 hour of the fermentation in presence of sucrose 4.46 U/mg of 572

3 protein and at the 7 th hour after cultivation of the mutant M2860 in media with glucose 1.65 U/mg of protein. The highest growth rate was reached to 8.95 g/l at the end of fermentation in media with sucrose. The final biomass concentration in media with glucose was lower (4.7 g/l) than that obtained on sucrose media. The highest growth rate was limited after ph 5.0, which led to inactivation of the synthesized GTFs from M2860. It is known that in a ph below 5.0, the metabolitic processes of Leuconostoc mesenteroides are direct to compensatory process of glucan synthesis when sucrose concentration is high or to reduction of fructose by the mannitol dehydrogenase into mannitol at low sucrose concentration (3, 17, 6, 10, 15). The profiles of the fermentation process after cultivation of M2860 in media with sucrose and glucose as a carbon source respectively, showed that the enzymes were secreted into the culture broth during growth. The maximum of glucose concentration was detected at the 3 th hour of cultivation in media with sucrose mmol/ml and in media with glucose mmol/ml (Table 1 and Table 2). The decrease of the level of glucose from 3 th to 5 th hour in the presence of sucrose and from 3 th to 6 th hour after cultivation of the mutant in media with glucose could be explained with its rapid metabolism that led to increase the level of detected products (acetate, lactate and ethanol) at the end of fermentations, and also with a transferase reaction of produced GTFs. The mutant M2860 produced more acetate than ethanol and lactate as in the presence of sucrose as glucose in ratios mmol/ml lactate : mmol/ml acetate : mmol/ml ethanol in the presence of sucrose, and mmol/ml lactate : mmol/ml acetate : mmol/ml ethanol in the presence of glucose (Table 1 andtable 2). In the presence of sucrose as a carbon source the maximum concentration of acetate was determined at the 6 th hour of cultivation mmol/ml, which was higher in comparison with the detected concentration of acetate in presence of glucose. A higher final concentration of detected acetate and lower concentrations of lactate and ethanol, produced by M2860 in media with sucrose could be explained with fructose metabolism via phosphoketolase pathway after phosphorilation. This is the reason for rapid acidification of fermentative medium and for the loss of GTF activity. It is known that GTFs are active in a ph range between (2). 573 TABLE 1 Dynamics of glucose, lactate, acetate and ethanol concentrations by cultivation of the mutant strain Leuc. mesenteroides M2860 in media containing 4% glucose Time (h) Glucose D/L- Lactate Acetate Ethanol TABLE 2 Dynamics of glucose, fructose, lactate, acetate and ethanol concentrations by cultivation of the mutant strain Leuc. mesenteroides M2860 in media containing 4% sucrose Time (h) Fructose Glucose D/L- Lactate Acetate Ethanol We studied the ratio between extracellular and cell associated GTFs from the parent strain Leuc. mesenteroides 28 and its constitutive mutant Leuc. mesenteroides M2860 after fermentation in media with sucrose (Fig. 3). It is known that some Leuc. mesenteroides strains produce extracellular and cell associated GTFs which synthesize different types of glucans (17, 20). Figure 3A showed that the parent strain produced mainly extracellular type GTF (91% of the total GTF activity). In contrast to Leuc. mesenteroides 28, much of the GTF activity (81%) in sucrose grown cultures of M2860 was located in the cell pellets (Fig. 3B). Probably the studied mutant strain produces more than one GTF and consequently different type of glucans than these synthesized by GTF from Leuc. mesenteroides 28. We did not detect GTF activity associated with the cells of M2860 after fermentation in media with glucose.

4 Leuc. mesenteroides 28 9% Gel B A 91% extracellular GTF (%) cell associated GTF (%) Leuc. mesenteroides M2860 Gel C 19% B 81% extracellular GTF (%) cell associated GTF (%) Fig. 3. Extracellular and cell associated GTF activities by a parent strain Leuc. mesenteroides 28 and a mutant strain M2860 To check if the mutant produces different type of GTFs when grown on glucose and sucrose, we separated extracellular and cell-associated proteins of sucrose- and glucose-containing media by SDS-PAGE, and polysaccharide-forming activity was detected in situ (Fig. 4). Fig. 4. In situ analysis of extracellular and cell associated GTFs from a parent strain Leuc. mesenteroides 28 and a mutant strain M2860 after fermentation in media containing 4% glucose and 4% sucrose. Gel А: 1 Cell associated GTF from a mutant М2860 after fermentation in media with 4% glucose; 2 Extracellular GTF from a mutant М2860 after fermentation in media with 4% glucose; 3 - Extracellular GTF from a parent strain 28 after fermentation in media with 4% sucrose; 4 Cell associated GTF from a parent strain 28 after fermentation in media with 4% sucrose; 5 Cell associated GTF from amutant М2860 after fermentation in media with 4% sucrose. Gel B: 1, 2 - Enzyme concentrate from a parent strain 28*; 3, 4 Enzyme concentrate from a mutant M2860**; 5 Extracellular GTFs from a mutant M2860*; 6 Enzyme concentrate from a mutant M2860*; * - after fermentation in media with 4% sucrose; ** - after fermentation in media with 4% glucose; Gel C: 1, 2 Extracellular GTF from a mutant М2860 after fermentation in media with 4% glucose; 3, 4 Extracellular GTF from a parent strain 28 after fermentation in media with 4% glucose. R Protein standards (1-myosin-220 kda, 2 - macroglobulin-170 kda, 3 - β- galactosidase-116 kda,4- transferin-76 kda, 5-glutamatdehydrogenase-53 kda) Gel A In situ analysis showed single band corresponding to 180 kda molecular size in a supernatant and enzyme concentrate obtained by fermentation of M2860 in media with glucose. The enzyme concentrate and the crude fraction obtained by fermentation of M2860 in media with sucrose showed a few molecular forms corresponding to 180 kda, 120 kda and 86 kda molecular sizes (Fig. 4, Gel B). The same bands were detected in the cell pellets of the mutant after fermentation in 574

5 media with sucrose (Fig. 4, Gel A). The concentrated enzyme and the supernatant fraction received after fermentation of the parent strain Leuc. mesenteroides 28 in media with sucrose showed a single band corresponding to 180 kda molecular size (Figure 4, Gel A, Gel B). In situ analysis showed no bands after cultivation of the parent strain Leuc. mesenteroides 28 in media with glucose (Fig. 4, Gel C). The proteins having molecular mass of 180 kda are corresponding to dextransucrase produced by strain Leuc. mesenteroides NRRL B-512F (2, 19, 5). Conclusions We showed that the mutant strain Leuc. mesenteroides M2860 produces detectable extracellular GTF activity with molecular mass corresponding to 180 kda when glucose replaces sucrose as a growth substrate. This confirms that the mutant strain Leuc. mesenteroides M2860 is constitutive for GTF production. Much of the GTF activity (81%) in sucrose grown cultures is located in the cell pellets. In situ analysis of the extracellular and cell associated enzymes from Leuc. mesenteroides M2860 after fermentation in media with sucrose showed three activity bands 180 kda, 120 kda and 86 kda. Acknowledgement This work was supported by research grant BG RNF of NSF Bulgaria and by DOO of NSF Bulgaria. REFERENCES 1. Bradford M. (1990) Analitical Biochemistry, 72, Dols, M., Remaud-Siméon, M., Monsan, P. (1997) Enzyme Microb. Technol., 20, Dols M., Remaud-Simeon M., Willemot R.M., Vignon M., Monsan P. (1998) Appl. Environ. Microbiol., 64, Du Bois M.A., Gilles K.A., Hamilton J.K., Rebers P.A., Smith F. (1956) Anal. Chem., 28, Goyal, A. and Katiyar, S. (1994) J. Microbiol. Methods, 20, Goyal A., and Katiyar S. (1997) J Basic Microbiol, 37(3), Iliev, I., Vassileva, T., Ignatova, C., Ivanova, I., Haertle, T., Monsan, P., Chobert, J-M. (2008) Journal of Applied Microbiology, 104, Iliev I., Ivanova, I., Remaud, M. and Monsan, P. (2003) Current Studies of Biotechnology, 3, Kim, D. and Robyt, J (1994) Enzime Microb. Technol., 16, Kitaoka, M. and Robyt, J. (1998) Enzime and Microbial Technol, 22, Laemmli U.K. (1970) Nature, 227, Miller A. W., Robyt J. (1986) Anal. Biochem, 156, Neely, W. and Not,t, J. (1962) Biochemistry, 1, Paul F., Monsan P., Auriol D. (1984) European patent Quirasco, M., Lopez-Munguia, A., Remaud-Simeon, M., Monsan, P., Farres, A. (1999) Applied and Environmental Microbiology, 65, Robyt, J. (1995) Adv Carbohydr Chem Biochem, 51, Robyt, J., Kim, D., Yu, L. (1995) Carbohydr. Res., 266, Smith, M. and Zahnley, J. (1997) Applied and Environmental Microbiology 63, Willemot, R., Monsan, P., Durand, G. (1988) Annals of the New York Academy of sciences, 542, Zahnley, J. and Smith, M. (1995) Appl. Environ. Microbiol., 61,