通过圆二色光谱研究在变性剂存在下里氏木霉内切葡聚糖酶的构象变化。

Study of conformational changes in glucoamylase of Aspergillus awamori nakazawa in presence of denaturants through CD-spectroscopy.

机构信息

Applied Mechanics Department, Motilal Nehru National Institute of Technology, Allahabad, Uttar Pradesh, India.

出版信息

Bioresour Technol. 2010 Oct;101(19):7577-80. doi: 10.1016/j.biortech.2010.04.042. Epub 2010 May 8.

DOI:10.1016/j.biortech.2010.04.042

PMID:20452763

Abstract

In present study, changes in the pattern or motifs of secondary structures of glucoamylase from Aspergillus awamori nakazawa in the presence of denaturants such as urea, guanidine-HCl (Gdn-HCl), and at different pH, were studied through CD-spectroscopy. It was observed that in native state glucoamylase entirely comprised of beta-sheets. CD-spectra of glucoamylase in the presence of 6 M urea lost its spectral regular characteristic; and ellipticity became zero between 207 and 218 nm. With 0.5 M urea concentration glucoamylase showed approximately double negative ellipticity between 207 and 218 nm than the control, which indicates increase in beta-sheets and decrease in random coil contents. But higher concentration of urea (>or=6 M) and Gdn-HCl (>or=3 M) completely unfold the enzyme. At lower concentration (0.1 M) of Gdn-HCl negative peak got shifted from 208 to 219 nm to a very sharp peak at 198 nm with lower intensity than the control. It was also observed that glucoamylase posses higher beta-sheet in acidic media than in basic media. Glucoamylase remains active in a broad range of pH (3.0-11.0) and maximum activity was observed at pH 4.5. Activity of glucoamylase does not vary too much between pH 5.5 and 9.0. Conformational changes during wide range of pH were supported with its activity coincided.

摘要

在本研究中，通过 CD 光谱研究了在变性剂如尿素、盐酸胍（Gdn-HCl）和不同 pH 值存在下，从 Aspergillus awamori nakazawa 中提取的葡糖淀粉酶的二级结构模式或基序的变化。结果表明，在天然状态下，葡糖淀粉酶完全由β-折叠组成。在 6 M 尿素存在下，葡糖淀粉酶的 CD 光谱失去了其光谱特征；在 207 至 218nm 之间，椭圆率变为零。在 0.5 M 尿素浓度下，葡糖淀粉酶在 207 至 218nm 之间的负椭圆率比对照约增加一倍，这表明β-折叠增加，无规卷曲含量减少。但更高浓度的尿素（≥6 M）和 Gdn-HCl（≥3 M）会使酶完全展开。在较低浓度（0.1 M）的 Gdn-HCl 下，负峰从 208nm 移至 219nm，在 198nm 处形成一个非常尖锐的峰，强度低于对照。还观察到葡糖淀粉酶在酸性介质中比在碱性介质中具有更高的β-折叠。葡糖淀粉酶在广泛的 pH 范围内（3.0-11.0）保持活性，在 pH 4.5 时具有最大活性。在 pH 5.5 和 9.0 之间，葡糖淀粉酶的活性变化不大。在广泛的 pH 值范围内发生的构象变化得到了其活性的支持，两者的变化是一致的。