Hydrolysis of galacto-oligosaccharides in soy molasses by α -galactosidases and invertase from Aspergillus terreus

719 Vol.53, n. 3: pp. 719-729, May-June 2010 ISSN 1516-8913 Printed in Brazil BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L Hydrolysis of Galacto-oligosaccharides in Soy Molasses by α-galactosidases and Invertase from Aspergillus terreus Angélica Pataro Reis, Valéria Monteze Guimarães, Joana Gasperazzo Ferreira, José Humberto de Queiroz, Maria Goreti Almeida Oliveira, Daniel Luciano Falkoski, Maíra Nicolau de Almeida and Sebastião Tavares de Rezende* 1 Departamento de Bioquímica e Biologia Molecular; Universidade Federal de Viçosa; 36570-000; Viçosa - MG Brasil ABSTRACT Two α-galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28°C were partially purified by gel filtration and hydrophobic interaction chromatographies. Optimum pH and temperatures for P1, P2 and invertase preparations were 4.5-5.0, 5.5 and 4.0 and 60, 55 and 65°C, respectively. The KM app for ρ-nitrophenyl-α-D-galactopyranoside were 1.32 mM and 0.72 mM for P1 and P2, respectively, while the KM app value for invertase, using sacarose as a substrate was 15.66 mM. Enzyme preparations P1 and P2 maintained their activities after pre-incubation for 3 h at 50°C and invertase maintained about 90% after 6 h at 55 °C. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses. Key words: Aspergillus terreus, galactooligosaccharides, α-galactosidase, invertase, soy molasses INTRODUCTION α-Galactosidase (α-D-galactoside galactohydrolase EC 3.2.1.22) catalyzes hydrolysis of terminal nonreducing α-D-galactose residues in oligosaccharides such as melibiose, raffinose and stachyose; polysaccharides, glycolipids and glycoproteins (Varbanets et al., 2001). This enzyme is widely distributed in microorganisms, plants, and animals (Dey et al., 1993). α-Galactosidases have a number of biotechnological applications. In the sugar beet industry these enzymes are used to remove raffinose from beet molasses and increase sucrose yields (Shibuya et al., 1995); they are also used to improve the gelling properties of galactomannans * (Bulpin et al., 1990) and to degrade galactooligosaccharides (GO) in food materials such as soy derivatives (Guimarães et al., 2001, Viana et al., 2005; Viana et al., 2007). The GO, especially raffinose and stachyose, are considered the major factors responsible for flatulence and intestinal disturbances in humans and monogastric animals after ingestion of soybeans and other legumes. Enzymatic hydrolysis of GO by α-galactosidases may be an alternative to improve the nutritional quality of soy products for animal and human consumption (de Rezende et al., 2005). α-Galactosidase has been purified from several microbial sources including Aspergillus ficcum (Zapater et al., 1990), Trichoderma reesei (Zeilinger et al., 1993) and A. oryzae (Prasshanth Author for correspondence: Braz. Arch. Biol. Technol. v.53 n.3: pp. 719-729, May/June 2010 720 Reis, A. P. et al and Mulimani, 2005), with multiple forms of α-galctosidase being reported (Luonteri et al., 1998; Manzanares et al., 1998; Ademark et al., 2001), Although, α-galactosidases are the main enzymes required for GO hydrolysis, and invertases can also be important to increase the hydrolysis yield, since GO are substrates for both the enzymes. The Aspergillus genus has been characterized as a good producer of several hydrolytic enzymes (de Rezende and Felix 1999; Noronha et al., 2002; Lemos et al., 2002; Souza-Mota et al., 2005). A. terreus, when grown on wheat straw, secreted multiple forms of α-galactosidase. One α-galactosidase form was partially purified and characterized, showing high hydrolysis GO potential in soy products (Falkoski et al., 2006). In the present study, the partial purification and characterization of new forms of α-galactosidases and invertase from A. terreus, aiming to elucidate their use in biotechnological applications was reported. MATERIALS AND METHODS Organism growth and enzyme production Aspergillus terreus CCT 4083 and Monascus rubber were obtained from the André Toselo Tropical Research Foundation, Campinas–SP, Brazil, and Aspegillus versicolor was obtained from the Bromatology Laboratory’s culture collection, DBB, Federal University of Viçosa, Viçosa, MG, Brazil. The stock cultures were maintained on potato dextrose agar media at 4°C. In order to select the carbon source, spores (107/mL) were transferred to 25 mL of liquid medium containing (in g/L) 7.0 KH2PO4, 2.0 K2HPO4, 0.1 MgSO4.7H2O, 1.0 (NH4)2SO4, 0.6 yeast extract, and 1% (w/v) of Dgalactose (Sigma Chemical Co., St. Louis, MO) or wheat straw. After incubation at 28°C, 150 rpm for 24-240 h, the culture supernatants were collected by filter paper filtration. The enzyme was produced in 5 L of the wheat straw medium. After 168 h of growth at 28 °C, the culture was filtered and the supernatants containing α-galactosidase activity were concentrated, approximately 10-fold, by lyophilization (de Rezende et al., 2005). Enzyme assay α-Galactosidase was assayed using ρ-nitrophenyl-α-D-galactopyranoside, ρNPGal, (Sigma) or other synthetic substrates, according to Falkoski et al., (2006). The reaction was carried out for 20 min at 40 °C, and ended by the addition of 1 mL 0.5 M Na2CO3. The amount of ρ-nitrophenol (ρNP) released was determined by spectrometry measurement at 410 nm. Invertase was assayed using sucrose, according de Rezende and Felix (1999). The reaction was carried out for 30 min at 40 °C using a reaction mixture containing 650 µL of 0.1 M sodium acetate buffer (pH 5.0) 100 µL of enzyme solution and 250 µL of sucrose. The amount of reducing sugar produced was determined according to Miller (1956).The activities against raffinose, stachyose, guar and locust bean gum were assayed for 30 min at 40 °C using a reaction mixture containing 650 µL of 0.1 M sodium acetate buffer (pH 5.0), 100 µL of enzyme solution and 250 µL of substrate solutions. The amount of reducing sugar produced was determined as above. The hydrolysis of melibiose, maltose, and lactose activities were evaluated by the glucose-oxidase method (Stemberg et al., 1970). Protein Concentration The protein concentration in the enzymatic samples was determined according to Bradford, (1976), with bovine serum albumin as the standard. α-Galactosidase purification The A. terreus culture supernatant was concentrated by lyophilization and chromatographed in a Sephacryl S-200 (Amersham Biosciences, Uppsala, Sweden) column (87.5 x 2.5 cm), equilibrated and eluted with 25 mM sodium acetate buffer, pH 5.0. Proteins were eluted at a flow rate of 20 mL/h and 4 mL fractions were collected. The active S2 protein fraction was pooled and concentrated using an Amicon ultrafiltration cell model 8400 (Bedford, MO) with a 10 kDa molecular cutoff membrane. The concentrated fraction was loaded in (...truncated)