Abstract

α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed K_m value of 2.3809 mM and V_max of 2.0 × 10⁴ µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

摘要

α-半乳糖苷酶水解存在于糖类非还原端的 α-1,6-键，导致从蜜二糖、棉子糖、水苏糖等低聚糖中释放半乳糖基残基。在本研究中，我们报告，α-来自弯曲芽孢杆菌的半乳糖苷酶从马尼卡兰温泉（印度）中分离出来。在 150 rpm 下 72 小时后，瓜尔胶和豆粕中的酶产量达到最大。同时，生产温度/pH 值分别优化为 50 °C 和 7.0。获得同工酶（α-gal I 和 II）并根据最佳温度/pH 及其稳定性特征进行表征。JS27 α-Gal II 的最终纯化倍数为 11.54。天然和 SDS-PAGE 用于确定酶的分子量分别为 86 和 41 kDa，表明其同源二聚体形式。JS27 α-Gal II 在 55 °C 和 pH 7（10 分钟）下表现出最佳酶活性。该酶的K _m值为 2.3809 mM，V _max为 2.0 × 10 ⁴ µmol/min/ml，以 pNPG 为底物。JS27 α-Gal II 展示了底物水解和转半乳糖基化产物 (α-GOS) 与多种底物（糖/糖醇、低聚糖和复合碳水化合物）的同时形成，这些通过 TLC 和 HPLC 分析得到验证。α-GOS 是重要的功能性食品成分，可以作为益生元进行探索。