Glycobiology and Protein Biochemistry Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Department of Plant Science, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
Int J Biol Macromol. 2021 Apr 1;175:558-571. doi: 10.1016/j.ijbiomac.2021.01.179. Epub 2021 Jan 30.
Alpha galactosidase is an exoglycosidase that cleaves α-D-galactose and has numerous applications in medicine, biotechnology, food and pharma industries. In this study, a low molecular weight acidic α-galactosidase was identified from the seeds of custard apple. The purification of α-galactosidase from the crude extract of defatted seeds was achieved by employing ammonium sulphate fractionation, hydrophobic interaction and gel filtration chromatographic techniques. The purified custard apple α-galactosidase (CaG) migrated as a single band in native PAGE corresponding to molecular weight of ~67 kDa and cleaved chromogenic, fluorogenic and natural substrates. CaG was found to be a heterodimer with subunit masses of 40 and 30 kDa. The kinetic parameters such as K and Vmax were found to be 0.67 mM and 1.5 U/mg respectively with p-nitrophenyl α-D-galactopyranoside. Galactose, methyl α-D-galactopyranoside and D-galacturonic acid inhibited CaG activity in mixed mode. The CD spectral analysis at far UV region showed that purified CaG exists predominantly as helix (35%), beta sheets (16.3%) and random coils (32.3%) in its secondary structure. These biochemical and biophysical properties of CaG provide leads to understand its primary sequence and glycan structures which will eventually define its novel physiological roles in plants and potential industrial applications.
α-半乳糖苷酶是一种外切糖苷酶，可切割α-D-半乳糖，在医学、生物技术、食品和制药行业有广泛的应用。在本研究中，从番荔枝种子中鉴定到一种低分子量酸性α-半乳糖苷酶。通过采用硫酸铵分级沉淀、疏水相互作用和凝胶过滤色谱技术，从脱脂种子粗提物中纯化α-半乳糖苷酶。纯化的番荔枝α-半乳糖苷酶（CaG）在天然聚丙烯酰胺凝胶电泳中迁移为单一带，对应的分子量约为 67 kDa，可切割显色、荧光和天然底物。CaG 被发现是一种异二聚体，亚基质量分别为 40 和 30 kDa。动力学参数如 K 和 Vmax 分别为 0.67 mM 和 1.5 U/mg，以对硝基苯-α-D-半乳糖吡喃糖苷为底物。半乳糖、甲基-α-D-半乳糖吡喃糖苷和 D-半乳糖酸以混合模式抑制 CaG 活性。远紫外区的 CD 光谱分析表明，纯化的 CaG 主要以螺旋（35%）、β 折叠（16.3%）和无规则卷曲（32.3%）的形式存在于其二级结构中。这些 CaG 的生化和生物物理性质为理解其一级序列和糖基结构提供了线索，这最终将确定其在植物中的新的生理作用和潜在的工业应用。
