Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru 570 020, India.
Department of Protein Chemistry and Technology, CSIR-Central Food Technological Research Institute, Mysuru 570 020, India; Academy of Scientific and Innovative Research (AcSIR), CSIR-CFTRI Campus, Mysuru, India.
Int J Biol Macromol. 2018 Nov;119:770-778. doi: 10.1016/j.ijbiomac.2018.06.093. Epub 2018 Jun 21.
A hitherto unknown low molecular weight form of α-galactosidase (VM-αGal-P) from germinating black gram (Vigna mungo) seeds was purified (324 U/mg specific activity, 1157-fold purification, ~45 kDa) using ion-exchange (DEAE-cellulose, CM-sepharose), gel filtration (Sephadex G-75) and affinity (Con-A Sepharose 4B) chromatography but with poor yield (0.75%). Partially purified enzyme (VM-αGal) (146.3 U/mg specific activity, 522.5-fold purification) was used for further studies. VM-αGal showed optimal activity at pH 5 and 55 °C. Hg and SDS completely inhibited VM-αGal activity. The K, V and catalytic efficiency (k/K) of VM-αGal for pNPG and raffinose was 0.99, 17.23 mM, 1.66, 0.146 μmol ml min, and 0.413, 0.0026 s mM, respectively. VM-αGal was competitively inhibited by galactose (K 7.70 mM). Thermodynamic parameters [activation enthalpy (ΔH), activation entropy (ΔS) and free energy (ΔG)] of VM-αGal at 45-51 °C showed that VM-αGal was in a less energetic state and had susceptibility towards denaturation. Temperature-induced structural unfolding studies of VM-αGal probed by fluorescence, and far-UV CD spectroscopy revealed significant loss in tertiary structure and a steep decline in β-sheet content at 45-65 °C, and above 55 °C, respectively. VM-αGal improved the nutritional quality of soymilk by hydrolyzing raffinose family oligosaccharides (26.5% and 18.45% decrease in stachyose and raffinose, respectively).
一种来自发芽黑大豆（Vigna mungo）种子的未知低分子量α-半乳糖苷酶（VM-αGal-P）经离子交换（DEAE-纤维素，CM-琼脂糖）、凝胶过滤（葡聚糖 G-75）和亲和（Con-A 琼脂糖 4B）层析纯化（324U/mg 比活性，1157 倍纯化，~45kDa），但收率低（0.75%）。部分纯化的酶（VM-αGal）（146.3U/mg 比活性，522.5 倍纯化）用于进一步研究。VM-αGal 在 pH 5 和 55°C 时表现出最佳活性。Hg 和 SDS 完全抑制了 VM-αGal 的活性。VM-αGal 对 pNPG 和棉子糖的 K、V 和催化效率（k/K）分别为 0.99、17.23mM、1.66、0.413μmol/ml/min 和 0.146μmol/ml/min、0.0026s mM。VM-αGal 被半乳糖竞争性抑制（K i 7.70mM）。VM-αGal 在 45-51°C 的热力学参数[活化焓（ΔH）、活化熵（ΔS）和自由能（ΔG）]表明，VM-αGal 处于能量较低的状态，易发生变性。荧光和远紫外 CD 光谱研究 VM-αGal 的热诱导结构展开表明，在 45-65°C 之间，VM-αGal 的三级结构明显丧失，β-折叠含量急剧下降，在 55°C 以上，分别。VM-αGal 通过水解棉子糖家族低聚糖（分别降低 26.5%和 18.45%的水苏糖和棉子糖）提高了豆浆的营养价值。
