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Purification of beta-galactosidase from Erythrina indica: Involvement of tryptophan in active site

机译:E虫中β-半乳糖苷酶的纯化:色氨酸参与活性位点

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摘要

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 Km 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 Zn2+ and Hg2+. 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% a helix, 27% beta sheet and 38% random coil in the secondary structure of the purified enzyme. (c) 2007 Elsevier B.V. All rights reserved.
机译:β-半乳糖苷酶(EC:3.2.1.23)是在ry桐种子中检测到的一种糖苷酶,被纯化至135倍。在四个主要的糖苷酶中,检测到的β-半乳糖苷酶被糖基化程度最低,并且未被Con-A CL浆液亲和基质保留。通过离子交换色谱,然后进行凝胶过滤,获得酶的均匀制剂。通过凝胶过滤和SDS-PAGE,发现该酶是分子量分别为74kDa和78kDa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,该酶的Km值为2.6 mM,V-max为3.86 U / mg,并被Zn2 +和Hg2 +抑制。酶活性受反馈抑制作用的调节,因为它被β-D-半乳糖抑制。化学修饰研究表明色氨酸和组氨酸参与了酶的活性。荧光研究也支持了色氨酸的参与,发现一个色氨酸存在于β-半乳糖苷酶的活性位点。圆二色性研究表明,纯化酶的二级结构中有37%的螺旋,27%的β折叠和38%的无规卷曲。 (c)2007 Elsevier B.V.保留所有权利。

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