Kestwal Rakesh M, Bhide Shobhana V
Division of Biochemistry, Department of Chemistry, University of Pune, Pune-411007, Maharashtra, India.
Biochim Biophys Acta. 2007 Oct;1770(10):1506-12. doi: 10.1016/j.bbagen.2007.07.002. Epub 2007 Jul 19.
beta-Galactosidase (EC: 3.2.1.23), one of the glycosidases detected in Erythrina indica seeds, was purified to 135 fold. Amongst the four major glycosidases detected beta-galactosidase was found to be least glycosylated, and was not retained by Con-A CL Seralose affinity matrix. A homogenous preparation of the enzyme was obtained by ion-exchange chromatography, followed by gel filtration. The enzyme was found to be a dimmer with a molecular weight of 74 kDa and 78 kDa, by gel filtration and SDS-PAGE, respectively. The optimum pH and optimum temperature for enzyme activity were 4.4 and 50 degrees C, respectively. The enzyme showed a K(m) value of 2.6 mM and V(max) of 3.86 U/mg for p-nitrophenyl-beta-D-galactopyranoside as substrate and was inhibited by Zn(2+) and Hg(2+). The enzyme activity was regulated by feed back inhibition as it was found to be inhibited by beta-D-galactose. Chemical modification studies revealed involvement of tryptophan and histidine for enzyme activity. Involvement of tryptophan was also supported by fluorescence studies and one tryptophan was found to be present in the active site of beta-galactosidase. Circular dichroism studies revealed 37% alpha helix, 27% beta sheet and 38% random coil in the secondary structure of the purified enzyme.
β-半乳糖苷酶（EC：3.2.1.23）是刺桐种子中检测到的糖苷酶之一，纯化了135倍。在检测到的四种主要糖苷酶中，β-半乳糖苷酶的糖基化程度最低，并且不被伴刀豆球蛋白A CL Seralose亲和基质保留。通过离子交换色谱，然后进行凝胶过滤，获得了该酶的纯制剂。通过凝胶过滤和SDS-PAGE分别发现该酶是分子量为74 kDa和78 kDa的二聚体。酶活性的最适pH和最适温度分别为4.4和50℃。以对硝基苯基-β-D-吡喃半乳糖苷为底物时，该酶的K(m)值为2.6 mM，V(max)为3.86 U/mg，并且受到Zn(2+)和Hg(2+)的抑制。该酶活性受反馈抑制调节，因为发现它受到β-D-半乳糖的抑制。化学修饰研究表明色氨酸和组氨酸参与酶活性。荧光研究也支持色氨酸的参与，并且在β-半乳糖苷酶的活性位点发现存在一个色氨酸。圆二色性研究表明纯化酶的二级结构中37%为α螺旋，27%为β折叠，38%为无规卷曲。
