Extraction and Biochemical Characterization of α-Galactosidase from Cajanus cajan Followed by Downstream Processing Using an Aqueous Two-Phase System (ATPS)

α-Galactosidase (EC 3.2.1.22) is an important biocatalyst with significant applications in food, feed, and biotechnological industries due to its ability to hydrolyze raffinose family oligosaccharides (RFOs) and synthesize functional oligosaccharides via transglycosylation. The present study focuses on the extraction, partial purification, biochemical characterization, and functional application of CC-α-Galactosidase from Cajanus cajan (Pigeon pea). The activity of CC-α-Gal was determined using p-nitrophenyl-α-D-galactopyranoside (p-NPGal) as substrate, supported by standard calibration curves of p-nitrophenol and bovine serum albumin for accurate quantification. Partial purification of CC-α-Gal was achieved through citric acid precipitation followed by ammonium sulfate fractionation (0–60%), resulting in a significant increase in specific activity from 0.06 to 1.68 U/mg with a 28-fold purification and 56.7% yield, indicating effective enrichment. Biochemical characterization revealed that CC-α-Gal exhibited maximum activity at pH 6.0 and 55 °C, suggesting optimal performance under slightly acidic and moderately elevated temperatures. CC-α-Gal showed high stability in the acidic pH range (4–6), while reduced stability was observed under alkaline conditions, indicating its suitability for applications in acidic environments.
Aqueous two-phase extraction (ATPS) using polyethylene glycol (PEG) and various salts was employed as a downstream processing strategy. Among the systems tested, PEG 3350 combined with ammonium sulfate demonstrated the highest recovery and purification efficiency of CC-α-Gal, highlighting the influence of PEG molecular weight and salt type on partitioning behavior. Furthermore, CC-α-Gal exhibited transglycosylation activity, enabling the synthesis of α-galacto-oligosaccharides (α-GOS) using melibiose (1.2 M) as both donor and acceptor substrate. Thin-layer chromatography (TLC) confirmed the time-dependent formation of α-GOS, while UPLC-ELSD analysis validated the presence of transglycosylation product manninotriose and another higher α-GOS alongside hydrolysis products
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(galactose/glucose). The transglycosylation results showed that the high substrate concentration favors transglycosylation over hydrolysis, demonstrating the potential of CC-α-Gal for producing α-GOS for the first time. In conclusion, CC-α-Gal from Cajanus cajan was successfully extracted, partially purified, and characterized, exhibiting desirable biochemical properties and significant transglycosylation potential. The study highlights its applicability in the removal of anti-nutritional factors and the synthesis of value-added prebiotic compounds, making CC-α-Gal a promising candidate for industrial applications.