Extraction and downstream processing of α-galactosidase from Pisum sativum using an aqueous two-phase system (ATPS).

α-Galactosidase (EC 3.2.1.22) is a key enzyme involved in the hydrolysis of raffinose family oligosaccharides (RFOs), which are widely present in leguminous seeds and are responsible for anti-nutritional effects in humans and monogastric animals. In the present study, α-galactosidase (PS-α-Gal) was extracted from Pisum sativum and subjected to partial purification, biochemical characterization, and evaluation of its transglycosylation potential for the synthesis of value-added oligosaccharides. The enzyme was initially obtained as a crude extract, yielding a total activity of 94.75 U and total protein content of 124 mg, with a specific activity of 0.76 U/mg. Partial purification was carried out using ammonium sulphate precipitation (0–60% saturation) followed by dialysis, resulting in an increase in specific activity to 1.21 U/mg with a purification fold of 1.59 and a recovery yield of 88.2%, indicating effective removal of non-specific proteins while retaining significant enzymatic activity.To improve purification efficiency, an aqueous two-phase system (ATPS) was employed using polyethylene glycol (PEG) of different molecular weights (PEG 3350, PEG 4600, and PEG 5000) in combination with various salts. The partitioning behavior of PS-α-Gal was strongly influenced by both PEG molecular weight and salt type. Among the systems evaluated, PEG 5000–sodium citrate showed the highest recovery (96%) and purification factor (2.82), with preferential partitioning of the enzyme into the salt-rich phase (K < 1). In contrast, PEG 4600 systems demonstrated superior purification efficiency, with PEG 4600–ammonium sulphate and PEG 4600–sodium sulphate yielding high purification factors of 5.3 and 5.19, respectively, along with significant enzyme recovery. PEG 3350 systems exhibited comparatively lower purification efficiency, indicating that higher molecular weight PEG favors improved enzyme partitioning and purification. These findings highlight the importance of optimizing phase components for effective enzyme recovery using ATPS.Biochemical characterization of PS-α-Gal revealed that the enzyme exhibits optimal catalytic activity at 55 °C, with a gradual increase in activity observed between 35 °C and 55 °C, followed by a decline at higher temperatures due to probable thermal denaturation. The enzyme showed maximum activity at pH 6, indicating preference for slightly acidic conditions. Stability
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studies demonstrated that PS-α-Gal retains high stability in the acidic pH range (pH 4–6), while significant loss of activity was observed at strongly acidic conditions (pH 3). The enzyme also exhibited appreciable stability under neutral and alkaline conditions, suggesting moderate structural tolerance to pH variations. These properties indicate its suitability for applications in food processing and biotechnology.The transglycosylation potential of PS-α-Gal was evaluated using melibiose (1.2 M) as both donor and acceptor substrate. Thin-layer chromatography (TLC) analysis confirmed the time-dependent formation of higher oligosaccharides, with increased product accumulation observed between 12 and 24 h. UPLC-ELSD analysis further validated the formation of transglycosylation products, with new peaks observed at retention times of 9.4 and 10.5 min, corresponding to higher degree oligosaccharides such as trisaccharides (DP3) manninotrioseand possibly tetrasaccharides (DP4). Simultaneous detection of monosaccharide peaks confirmed the coexistence of hydrolysis and transglycosylation reactions, although the latter was favored under high substrate concentration conditions.Overall, this study demonstrates that PS-α-Gal from Pisum sativum is an efficient enzyme for both hydrolysis of anti-nutritional oligosaccharides and synthesis of functional α-galacto-oligosaccharides. The optimized ATPS system provides an effective strategy for enzyme recovery and purification, while the biochemical properties and transglycosylation capability highlight its potential application in prebiotic production and food industry processes