TRANSGALACTOSYLATION FOR GALACTOOLIGOSACCHARIDE SYNTHESIS USING PURIFIED AND CHARACTERIZED RECOMBINANT α-GALACTOSIDASE FROM Aspergillus fumigatus IMI 385708 OVEREXPRESSED IN Aspergillus sojae

Galactooligosaccharides are well-known functional food ingredients with prebiotic properties. Recent trend for the use of galactooligosaccharides in the food industry leads the search for new enzymes for their production. α-Galactosidase from Aspergillus fumigatus IMI 385708, possessing a highly efficient debranching ability on polymeric substrates, is also able to perform transgalactosylation. In this study, recombinant α-galactosidase produced by Aspergillus sojae Ta1 was purified 18.7-fold using anion exchange and hydrophobic interaction chromatography with an overall yield of 56% and 64.7 U/mg protein specific activity. The Vmax and Km values for the hydrolysis of pNPGal were 78 U/mg protein and 0.45 mM, respectively. Optimum pH (pH 4.5) and temperatures (50- 60°C) for recombinant α-galactosidase activity were determined. For the synthesis of oligosaccharides, purified and characterized recombinant α-galactosidase was used in the transgalactosylation of various mono- and disaccharides using pNPGal (p-nitrophenyl-α-D-galactopyranoside) as galactose donor. Di- and trisaccharides obtained by transgalactosylation were analysed by TLC, ESI-MS, and HPLC analysis. Among 12 acceptor candidates, α-galactosidase transgalactosylated galactose, glucose, mannose, cellobiose, lactose, maltose, and sucrose efficiently, however, did not transgalactosylate xylose, arabinose, fucose, fructose, and melibiose.

PDF

___

Cobucci-Ponzano, B., Conte, F., Bedini, E., Corsaro, M.M., Parrilli, M., Sulzenbacher, G., Lipski, A., Dal Piaz, F., Lepore, L., Rossi, M. (2009). β-Glycosyl azides as substrates for α-glycosynthases: Preparation of efficient αL-fucosynthases. Chemistry & Biology, 16, 1097-1108.

Cobucci-Ponzano, B., Zorzetti, C., Strazzulli, A., Carillo, S., Bedini, E., Corsaro, M.M., Comfort, D.A., Kelly, R.M., Rossi, M., Moracci, M. (2011). A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-Dgalactopyranosyl azide to alpha-galacto-oligosaccharides. Glycobiology, 21, 448-456.

Crout, D.H.G., Vic, G. (1998). Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Current Opinion in Chemical Biology, 2, 96-111.

Dey, P.M., Pridham, J.B. (1972). Biochemistry of α-galactosidases. Advanced Enzymology, 36, 91-130.

Dey, P.M., Patel, S., Brownleader, M.D. (1993). Induction of alpha-galactosidase in Penicillium ochrochloron by guar (Cyamopsis tetragonobola) gum. Biotechnology and Applied Biochemistry, 17, 361-371.

Duffaud, G.D., McCutchen, C.M., Leduc, P., Parker, K.N., Kelly, R.M. (1997). Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068. Applied and Environmental Microbiology, 63, 169-177.

Gibson, G.R., Roberfroid, M.B. (1995). Dietary Modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition, 125, 1401-1412.

Gupta, R., Jung, E., Brunak, S. (2004). Prediction of N-glycosylation sites in human proteins. http://www.cbs.dtu.dk/services/NetNGlyc/

Gurkok, S., Soyler, B., Biely, P., Ogel, Z.B. (2010). Cloning and heterologous expression of the extracellular alphagalactosidase from Aspergillus fumigatus in Aspergillus sojae under the control of gpdA promoter. Journal of Molecular Catalysis B: Enzymatic, 64, 146-149.

Gurkok, S., Cekmecelioglu, D., Ogel, Z.B. (2011). Optimization of culture conditions for Aspergillus sojae expressing an Aspergillus fumigatus α-galactosidase. Bioresource Technology, 102, 4925-4929.

Hashimoto, H., Katayama, C., Goto, M., Okinaga, T., Kitahata, S. (1995). Transgalactosylation catalyzed by α-galactosidase from Candida guilliermondii H-404. Bioscience, Biotechnology, and Biochemistry, 59(4), 619-623.

Hinz, S.W.A., Doeswijk-Voragen, C.H.L., Schipperus, R., Broek, L.A.M., Vincken, J.P., Voragen, A.G.J. (2005). Increasing the transglycosylation activity of α-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis. Biotechnology and Bioengineering, 93(1), 122-131.

Homann, A., Seibel, J. (2009). Towards tailor-made oligosaccharides-chemo-enzymatic approaches by enzyme and substrate engineering. Applied Microbiology and Biotechnology. 83, 209-216.

Honda, Y., Kitaoka, M. (2006). The first glycosynthase derived from an inverting glycoside hydrolase. Journal of Biological Chemistry, 281, 1426-1431.

Katrolia, P., Rajashekhara, E., Yan, Q., Jiang, Z. (2014). Biotechnological potential of microbial α-galactosidases. Critical Reviews in Biotechnology, 34, 307-317.

Kotwal, S.M., Gote, M.M., Sainkar, S.R., Khan, M.I. & Khire, J.M. (1998). Production of α-galactosidase by thermophilic fungus Humicola sp. in solid-state fermentation and its application in soyamilk hydrolysis, Process Biochemistry, 33, 337-343.

Kurt, F., 2011. Biotransformations in Organic Chemistry: A Textbook, 6th Eddition, Springer, Berlin. ISBN 978-3- 642-17393-6

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.

Liebl, W., Wagner, B., Schellhase, J. (1998). Properties of an a-galactosidase, and structure of its gene, galA, within an a- and ß-galactoside utilization gene cluster of the hyperthermophilic bacterium Thermotoga maritima. Systematic and Applied Microbiology, 21, 1-11.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.

Mackenzie, L.F., Wan, Q.P., Warren, R.A.J., Withers, S.G. (1998). Glycosynthases: Mutant glycosidases for oligosaccharide synthesis. Journal of the American Chemical Society, 120, 5583-5584.

Mi, S., Meng, K., Wang, Y., Bai, Y., Yuan, T., Luo, H., Yao, B. (2007). Molecular cloning and characterization of a novel α-galactosidase gene from Penicillium sp. F63 CGMCC 1669 and expression in Pichia pastoris. Enzyme and Microbial Technology, 40, 1373-1380.

Miyasato, M., Ajisaka, K. (2004). Regioreselectivity in beta-galactosidase-catalyzed transglycosylation for the enzymatic assembly of D-galactosyl-D- mannose. Bioscience, Biotechnology, and Biochemistry, 68, 2086- 2090.

Moracci, M., Trincone, A., Perugino, G., Ciaramella, M., Rossi, M. (1998). Restoration of the activity of activesite mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles. Biochemistry, 37, 17262-17270.

Neustroev, K.N., Krylov, A.S., Abroskina, O.N., Firsov, L.M., Nasonov, V.V., Khorlin, A.Y., (1991). Isolation and properties of α-galactosidase from Aspergillus awamori. Biochemistry USSR, 56, 288-296.

Perugino, G., Trincone, A., Rossi, M., Moracci, M. (2004). Oligosaccharide synthesis by glycosynthases. Trends in Biotechnology, 22, 31-37.

Puchart, V., Vrsanska, M., Bhat, M.K., Biely, P. (2000). Purification and characterization of α-galactosidase from a thermophilic fungus Thermomyces lanuginosus. Biochimica et Biophysica Acta, 1524, 27-37.

Puchart, V., Biely, P. (2005). Glycosylation of internal sugar residues of oligosaccharides catalyzed by α-galactosidase from Aspergillus fumigatus. Biochimica et Biophysica Acta, 1726, 206-216.

Rezessy-Szabó, J.M., Nguyen, Q.D., Hoschke, A., Braet, C., Hajós, G., Claeyssens, M. (2007). A novel thermostable α-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: Purification and characterization. Biochimica et Biophysica Acta, 1770, 55- 62.

Shaikh, F.A., Withers, S.G., (2008). Teaching old enzymes new tricks: Engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis. Biochemistry and Cell Biology, 86, 169-177.

Usui, T., Morimoto, S., Hayakawa, Y., Kawaguchi, M., Murata, T., Matahira, Y. (1996). Regioselectivity of beta-Dgalactosyl-disaccharide formation using the beta-D-galactosidase from Bacillus circulans. Carbohydrate Research, 285, 29-39.

Van Laere, K.M.J., Hartemink, R., Beldman, G., Pitson, S., Dijkema, C., Schols, H.A., Voragen A.G.J. (1999). Hydrolase and transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase. Applied Microbiology and Biotechnology, 52(5), 681-688.

Wada, J., Honda, Y., Nagae, M., Kato, R., Wakatsuki, S., Katayama, T., Taniguchi, H., Kumagai, H., Kitaoka, M., Yamamoto, K. (2008). 1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism. FEBS Letters, 582(27), 3739-3743.

Wang, H., Ma, R., Shi, P., Xue, X., Luo, H., Huang, H., Bai, Y., Yang, P., Yao, B. (2014). A new α-galactosidase from thermoacidophilic Alicyclobacillus sp. A4 with wide acceptor specificity for transglycosylation. Applied Biochemistry and Biotechnology, 174, 328-338.

Yamashita, A., Hashimoto, H., Fujita, K., Okada, M., Mori, S., Kitahata S. (2005). Reverse reaction of Aspergillus niger APC-9319 α-galactosidase in a supersaturated substrate solution: production of α-linked galactooligosaccharide (α-GOS). Bioscience, Biotechnology, and Biochemistry, 69(7), 1381-1388.

Zapater, I.G., Ullah, A.H.J., Wodzinsky, R.J. (1990). Extracellular alpha-galactosidase (E.C. 3.2.1.22) from Aspergillus ficuum NRRL 3135 purification and characterization. Preparative Biochemistry, 20, 263-296.