Preparation and characterization of cross-linked immobilized α-galactosidase enzyme aggregates and investigation of the potential use of them for the hydrolysis processes of raffinose and raffinose type oligosaccharides

α-D-Galactosidases (α-D-galactoside galactohydrolase, Mellibiase, EC. 3.2.1.22) are exoglycosidases that catalyse the hydrolysis of α-1,6-bounded D-galactose residues of basic and complex oligo- and polysaccharides (raffinose, stachyose, mellibiose, galactomannans). They are widely distributed in nature and found in plants, animals and microorganisms. α-Galactosidases are also involved in transgalactosylation reactions of α-galactosidic linkages of galactose conjugates. Both the hydrolase and transferase activity of α-galactosidases are very important for their industrial applications. They have many potential biotechnological and medicinal applications such as in food processing and animal feed processing, sugar industry, pulp and paper industry, enzymatic synthesis, structural analysis, conversion of blood type and treatment of Fabry' s disease. Since enzyme immobilization technology is one of the main methods for enzyme stabilization, it has become very important in recent years. In this context, enzyme immobilization methods that can be used in enzyme immobilization technology are increasing day by day. In recent years, the enzyme immobilization technique, prepared with Cross-Linked Enzyme Aggregates (CLEAs) in enzyme immobilization, is quite popular. There is no need for a carrier for immobilization. Generally, proteins of sufficient purity are cross-linked with bifunctional reagents to form cross-linked enzyme aggregates. CLEA's are very active enzyme preparations as well as good mechanical properties. Because they do not contain very high levels of foreign material that do not contain enzymatic activity and they can show increased stability even under extreme conditions, which makes their practice even more valuable. An important advantage of CLEA is that the protein used during immobilization does not need to be very pure, and crude enzyme preparations can be used. In this study, cross-linked enzyme aggregates (CLEA) of α-galactosidase enzyme isolated and partially purified from maize (Zea mays) were prepared and immobilized. The effects of various parameters (ammonium sulfate concentration, protein amount, glutaraldehyde concentration, buffer type, concentration and pH, cross-linking time, temperature and rate, BSA addition) were examined for immobilization for the optimization of the immobilization procedure used. CLEA was prepared with 47% recovery of activity at optimum conditions (1:5 (v/v) enzyme:ammonium sulfate ratio, 7.5 mg protein and 0.1% (v/v) glutaraldehyde, 6 hours, 4°Ϲ, 150 rpm). Physical and chemical characterization of prepared immobilized and free enzymes was carried out. Stability tests (thermal, pH, storage, operational stability) and reusability were performed by examining some parameters affecting enzyme activity (temperature, pH, substrate specificity and concentration, effector concentrations, inhibition effect, stabilizer effect). Immobilized and free enzymes showed maximum activity at 40 °C and their optimal pH values were pH 5.5 and 6.0, respectively. Kinetic constants (kM, vmax and kcat) were determined for free and immobilized enzymes in the presence of different substrates (pNPG, stachyose, melibiose and raffinose). The effects of various effectors on enzyme activity were investigated. The thermal, pH, storage and operational stability of α-Galactosidase CLEA was found to be quite good compared to the free enzyme. Studies on the potential use of free and immobilized α-galactosidase enzymes in hydrolysis processes of raffinose and raffinose-type oligosaccharides in soy milk have been carried out. CLEA and free enzyme hydrolyzed more than 85% of raffinose and stachyose in soy milk.