Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles
Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles
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DOI：
10.1016/j.bbapap.2006.03.016
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发表时间：
2006-06-01
影响因子：
3.2
通讯作者：
Halling, Peter J.
中科院分区：
文献类型：
--
作者：
Ganesan, Ashok;Price, Nicholas C.;Halling, Peter J.
固定化酶在工业上应用广泛，但这种生物催化剂失去活性的原因通常不清楚。我们用圆二色谱(CD)研究了一个这样的体系的结构，即固定在硅胶颗粒上的枯草杆菌嘉士伯(SC)(60 Pm)。必须克服一些技术问题，以便获得可得出结论的适当数据。为了避免在光谱采集过程中二氧化硅颗粒的沉积，已经开发了一种旋转式电池固定器。通过将电池固定器移动到尽可能靠近检测器窗口的位置，可以将差分散射的影响最小化。然而，吸收平坦化的影响限制了从远紫外CD研究中获得关于二级结构含量的可靠定量信息的程度。我们使用了一种基于高压产生的吸光度单位的经验方法来修正吸收平坦化效应。修正后的溶液光谱与实验结果吻合较好。有机介质中新鲜的和使用过的(不活跃的)SC-硅胶光谱的比较显示二级结构发生了实质性的变化。近紫外CD光谱的显著下降为天然构象的丧失提供了额外的证据。这些结果首次清楚地证明了酶在固定化状态下不稳定的根源。(C)2006爱思唯尔B.V.保留所有权利。
Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 pm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state. (c) 2006 Elsevier B.V. All rights reserved.