ENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES AND ALKYLGLYCOSIDES IN WATER ORGANIC MEDIA VIA TRANSGLYCOSYLATION OF LACTOSE

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1 ENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES AND ALKYLGLYCOSIDES IN WATER ORGANIC MEDIA VIA TRANSGLYCOSYLATION OF LACTOSE E. Bankova, N. Bakalova, S. Petrova, D. Kolev Sofia University St. Kliment Ohridski, Faculty of Biology, Department of Biochemistry, Sofia, Bulgaria ABSTRACT The synthesis of oligosaccharides and alkylglycosides via transglycosylation reaction of lactose by Aspergillus oryzae β-glycosidase was studied in the presence of water miscible organic solvents - DMSO (N, N -dimethylsulfoxid) and DMF (N, N - dimethylformamide) and water immiscible hydrophilic alcohols - isopropanol (2-propanol), secondary butanol (2-butanol) and isobutanol (2-metyl-1-propanol). In the presence of DMSO and DMF, both the transglycosylation and hydrolytic activities of the enzyme were suppressed. The yield of trisaccharides after 24h incubation was about 5% in the presence of % DMSO or % DMF. In a medium containing secondary butanol no transglycosylation products were detected. When isopropanol (%) was used, trisaccharides (3.54%) and isopropylgalacosides (7.52%) were obtained after 24h incubation. The highest transglycosylation activity of Aspergillus oryzae β-glycosidase was demonstrated in the presence of isobutanol. Increase in isobutanol concentration (up to 5%) enhanced the amount of transglycosylation products and a maximum yield of 6.68% trisaccharides and 14.5% isobutylgalactosides was reached. Introduction For several years, enzymatic synthesis in water-organic media has been of considerable interest (1, 2). Most of the enzymes used in such media are proteases, lipases and oxidases (3). Comparatively few studies have been performed on glycosidases. In water-organic media glycosidases have potential applications for synthesis of oligosaccharides and alkylglycosides (4, 5). Oligosaccharides in the form of glycoconjugates have a range of important functions in the biological systems, including fertilization, embryogenesis, cell proliferation, signal transduction, and therefore have potential as therapeutic agents (6). Moreover some galacto-oligosaccharides, such as trimmers and larger oligomers of saccharides (the enzymatic transgalactosylation products from lactose), were found Biotechnol. & Biotechnol. Eq. /6/3 114 to be good growing factors for intestinal Bifidobacteria (7) and therefore are of great interest for human health (8, 9). Alkylglycosides are a class of non-ionic surfactants and have several interesting properties in detergency, foaming, wetting, emulsification, and antimicrobial effect (1). Alkylglycosides are nontoxic, nonskin irritating, and very biodegradable. For these reasons, they have potential application in pharmaceutical, chemical, cosmetic, food, and detergent industries (11). Chemical synthesis of oligosaccharides and alkylglycosides usually requires selective protection and deprotection of the hydroxyl groups of the carbohydrates and activation of the anomeric carbon. In contrast to chemical synthesis, enzymatic approach (by using glycosidases) can provide regio- and stereo-selective products without

2 any protection or activation in a one step reaction (12). In our previous studies, the conditions for oligosaccharides production by Aspergillus oryzae β-glycosidase via transglycosylation of lactose in water reaction system, were optimized. It has been established that the enzyme transfers galactose to lactose at ph 5.5, 4 C, 65 IU (International Units), 15% lactose, and 24h incubation. Under these optimal conditions, 11.% of disaccharides, 11.4% of trisaccharides and only 2.4% of tetrasacharides were produced (13). The aim of the present work is to study the synthesis of oligosaccharides and alkylglycosides via transglycosylation reaction of lactose by Aspergillus oryzae β-glycosidase in the presence of water miscible organic solvents, such as DMSO and DMF, and water immiscible hydrophilic alcohols - isopropanol, secondary butanol, and isobutanol. Materials and Methods Enzyme The commercially available polyenzyme product Luizym from Aspergillus oryzae was provided by Luitpold-Pharma GmbH (Munich, Germany). β-glycosidase activity was purified through different chromatographic procedures, including gel-filtration on Acrylex P-1 (XK 2.6/1), anionexchange chromatography on Mono Q HR 5/5 and molecular-sieve chromatography on Superose 12 HR 1/3 (13). The purified protein fraction showed high β-galactosidase and transglycosylation activities and a single protein band on SDS-PAGE (sodium dodecyl sulfate-polyacryl amid gel electrophoresis). This partially purified β-glycosidase (6.81 fold) with a recovery of 34.72% and specific β-galactosidase activity of 5562 IU/g protein was used for further investigations (13). Materials The following reagents were used in the study: onpgal (o-nitrophenyl-β-d-galactopyranoside), DMSO (N, N - dimethylsulfoxide), DMF (N, N - dimethylformamide) - provided by Sigma Chemical Co. (St. Luis, Mo., USA); DEAE (diethyl amino ethyl)-si-1 4.5/25 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden); lactose was a product of Fluka Chemie AG (Germany); isobutanol (2-metyl-1-propanol), isopropanol (2-propanol), secondary butanol (2-butanol) were purchased from Serva Gmbh (Heidelberg, Germany). Enzyme activity assay β-galactosidase activity was determined by measuring the rate of hydrolysis of onpgal (14). The reaction mixture containing.9 ml of 2 mm substrate in.1 M sodium acetate buffer, (ph 4.8) or various organic solvents (-% v/v) and.1 ml appropriately diluted enzyme was incubated at 4 C for min. The reaction was terminated and color developed after the addition of.5 ml of 1M Na 2 CO 3. The amount of o- nitrophenol liberated was monitored at 45 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the release of 1 μmol o-nitrophenol per min under the assay conditions. Protein content estimation The protein concentration was determined according to Lowry et al (15). Transglycosylation activity The reaction mixtures containing 15% lactose, 5 mm sodium phosphate buffer (ph 5.5), 65 IU enzyme and various organic solvents (-5% v/v) in a total volume of ml, were incubated at 4 ºC. The aliquots were withdrawn at different time intervals during the period of 24h and the amount of products was determined by HPLC (High Performance Liquid Chromatography). Products analyses The amount of transglycosylation products was determined by HPLC (Waters-Millipore), using DEAE-Si-1 4.5/25 column with acetonitrile:water (: v/v) as a mobile phase at a flow rate of 1 ml/min. The elution was detected by differential refractometer (R 41, Millipore, USA). The dis- 115 Biotechnol. & Biotechnol. Eq. /6/3

3 Fig. 1. HPLC analyses of the products formed from lactose after 24h incubation in media containing % miscible organic solvents: A) DMSO; B) DMF; 1-disaccharides, 2-lactose, 3-trisaccharides. tribution and yield of each product were calculated by peak areas (HPLC analyses) and expressed as a percentage from the total sugar content. Results and Discussion Transglycosylation reaction in water organic media containing miscible organic solvents Water causes undesirable secondary hydrolysis and reduces the yield of oligosaccharides. For these reasons, in our studies we used miscible organic solvents (-5% v/v DMSO or DMF) in the reaction mixtures, in order to decrease water activity (a w ) and enhance transglycosylation ability of the enzyme. In contrast to the results of other authors (16), we found out that, in the presence of DMSO and DMF, both the transglycosylation and hydrolytic activity of Aspergillus oryzae β-glycosidase were suppressed. The maximum yield of trisaccharides in % DMSO or % DMF was about 5% (at optimal conditions for transglycosylation - ph 5.5, 4 C, 65 IU, 15% lactose, 24h incubation) and was lower then, the one obtained in water medium -11.4%. In such media, disaccharides and tetrasaccharides were not detected (Fig. 1). In the reaction media, containing 3% DMSO or 3% DMF, no transglycosylation products were found and the amount of hydrolyzed lactose was only about 1%. The most possible reason for these results is the inactivation of the enzyme caused by the lowering of the dielectric constant in the presence of miscible organic solvents such as DMSO (dielectric constant - ε=38.3, partition coefficient - Log P= ) and DMF (ε=47.2, Log P= -1.1). Under these conditions, the electrostatic interactions between the polar and charged residues are increased, thus reducing the protein flexibility and the accessibility of the substrates to the active site (17). Another possible reason for the enzyme inactivation could be the ph change that occurs in the presence of charged solvents, such as DMSO and DMF. Transglycosylation reaction in water organic media containing immiscible hydrophilic alcohols Immiscible hydrophilic alcohols (secondary Biotechnol. & Biotechnol. Eq. /6/3 116

4 Product distribution (%) lactose isopropylgalactosides monosaccharides trisaccharides Time (h) Fig. 2. Time course of oligosaccharide and isopropylgalactosides synthesis by Aspergillus oryzae β-glycosidase. Reaction mixture: % isopropanol, 15% lactose, 5mM sodium phosphate buffer (ph 5.5), and 65 IU of enzyme in a total volume of ml; T - 4 ºC. butanol, isopropanol and isobutanol) were used in reaction media as glycosyl acceptor for alkylglycoside synthesis via transglycosylation of lactose (glycosyl donor). Secondary butanol as acceptor In the presence of secondary butanol (ε=15.8, Log P=.61) as acceptor, no transglycosylation products were detected. Even in small concentrations (% v/v), secondary butanol causes complete inhibition of transglycosylation reaction. The hydrolysis of lactose was also low (after 24h incubation only 2% of the initial lactose was hydrolyzed). Among all studied organic solvents, secondary butanol was found to be the strongest inhibitor of the enzyme. The inactivation of the β-glycosidase in this case could be a result of the stripping effect of the alcohol (hydrophilic organic solvents can displace water molecules forming a layer around the protein, thus changing the enzyme conformation) (18). Moreover, interfacial inactivation of biocatalyst occurs in biphasic systems. Isopropanol as acceptor When % of isopropanol (ε= 18.3, Log P=.5) was used as glycosyl acceptor in the reaction mixture, under optimal conditions for transglycosylation (ph 5.5, 4 C, Product distribution (%) Isopropanol % (v/v) trisaccharides isopropylgalactosides monosaccharides lactose Fig. 3. Distribution of products formed from lactose by Aspergillus oryzae β-glycosidase after 24h incubation in media containing isopropanol as acceptor. Reaction mixtures: 15% lactose, 5mM sodium phosphate buffer (ph 5.5), 65 IU of enzyme and - 4% (v/v) isopropanol in a total volume of ml; T - 4 ºC. 65 IU, 15% lactose), trisaccharides and isopropylgalacosides were obtained. An increase in the amount of transglycosylation products (trisaccharides and alkyl glycosides) was observed after 2h incubation. The maximum yield of trisaccharides (3.54%) and isopropyl-galacosides (7.52%) was reached at 24h (Fig. 2). These results show that the use of small isopropanol con- 117 Biotechnol. & Biotechnol. Eq. /6/3

5 Product distribution (%) monosaccharides isobutylgalactosides lactose trisacchadides Time (h) Fig. 4. Time course of oligosaccharide and isobutylgalactosides synthesis by Aspergillus oryzae β-glycosidase. Reaction mixture: 5% isopropanol, 15% lactose, 5mM sodium phosphate buffer (ph 5.5), and 65 IU of enzyme in a total volume of ml; T - 4 ºC. centrations (up to %) is suitable for oligosaccharide synthesis, although some extent of β-galactosidase activity inactivation was detected (19). Increase in isopropanol concentration decreases both transglycosylation and hydrolytic activity of the enzyme. In reaction media containing organic solvent higher than % the yield of isopropyl galactosides was very low and oligosaccharides were not detected (Fig. 3). Isobutanol as acceptor The enzyme showed highest transglycosylation activity in the presence of isobutanol (ε= 18.7, Log P=.74). The transglycosylation was initiated at 2h and the maximum yield of trisaccharides (6.68%) and isobutylgalactosides (14.5%) was obtained after 24h of incubation (Fig. 4). Increase in isobutanol concentration (up to 5%) enhanced the amount of both hydrolysis and transglycosylation products (Fig. 5). The high hydrolytic and transglycosylation activities in all media containing isobutanol were surprising because isobutanol is a hydrophilic solvent (log P < 2), although compared to other studied organic solvents it has the highest value for Log P. These results show that in the case of isobutanolwater mixtures (low dielectric constant and low log P), the solvent is able to maintain the active catalytic conformation of the enzyme. Additional studies on the structure of the purified β-galactosidase should be made in order to elucidate enzyme solvent interactions. Conclusions The results presented show that Aspergillus oryzae β-glycosidase with high transglycosylation activity could be used for oligosaccharide synthesis not only in water medium, but also in media containing organic solvents. Isobutanol is the most appropriate solvent for the synthesis of alkylglycosides via transglycosylation of lactose. The use of small isopropanol concentrations is also suitable for the synthesis of isopropylgalactosides. The products synthesized have potential application in pharmaceutical, chemical, cosmetic, food, and detergent industries. REFERENCES 1. Vermuё M., Tramper J. (1995) Pure & Appl. Chem., 67, Gupta M., Roy I. (4) Eur. J. Biochem., 271, Biotechnol. & Biotechnol. Eq. /6/3 118

6 Product distribution (%) trisaccharides Isobutanol % (v/v) isobutylgalactosides monosaccharides lactose Fig. 5. Distribution of products formed from lactose by Aspergillus oryzae β-glycosidase after 24h incubation in media containing isobutanol as acceptor. Reaction mixtures: 15% lactose, 5mM sodium phosphate buffer (ph 5.5), 65 IU of enzyme, and -6% (v/v) isobutanol in a total volume of ml; T - 4 ºC. 3. Vulfson E., Halling P., Holland H. (1) Method in Biotehnol., 15, Enzyme in nonaqueous solvents: Methods and Protocols. Humana press Inc, New Jersey., Priya K., Loganathan D. (1999) Tetrahedron, 55, Vic G., Thomas D., Crout D. (1997) Enz. Microbial Technol.,, Perugino G., Trincone A., Rossi M., Moracci M. (4) Trend in Biotechnol., 22, Rabiu B., Jay J., Gibson G., Rastall R., (1) Appl. Environ. Microbiol., 67, Onishi N., Yamashiro A., Yokozeki K. (1995) Appl. Environ. Microbiol., 61, Hughes D., Hoover D. (1991) Food Technol., 45, Ismail A., Soultani S., Choul M. (1999) J. Biotechnol., 69, Rantwijk F., Oosterom, M., Sheldon R. (1999) J. of Mol. Cat., 6, Singh S., Scigelova M., Vic G., Crout D., (1996) J. Chem. Soc. Perkin Trans., 1, Petrova S., Bakalova N., Bankova E., Andonova S., Kolev D. (2) Pharmazie, 58, Wood T., Bhat K., (1988) In: Methods Enzymol., 16, Lowry O., Rosebrough N., Farr A, Randall R., (1951) J. Biol.Chem., 193, Fan J., Takegawa K., Iwahara S., Kondo A., Kato I., Abeygunawardana C., Lee Y. (1995) J. Biolog. Chem., 27, Girard E., Legoy D. (1999) Enzym Microl. Technol., 24, Gorman L., Dordick J. (1992) Boitechnol. Bioeng., 39, Bankova E., Petrova S., Bakalova N. (4) Ann. de l Université de Sofia, 96, Biotechnol. & Biotechnol. Eq. /6/3