Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi County 60004, Taiwan.
Protein J. 2010 Nov;29(8):572-82. doi: 10.1007/s10930-010-9287-8.
Environmental variables can significantly influence the folding and stability of a protein molecule. In the present study, the biophysical properties of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC) were characterized in detail by glutaraldehyde cross-linking, analytical ultracentrifugation, and various spectroscopic techniques. With cross-linking experiment and analytical ultracentrifuge, we demonstrated that the oligomeric state of BACΔNC in solution is monomeric. Far-UV circular dichroism analysis revealed that the secondary structures of BACΔNC were significantly altered in the presence of various metal ions and SDS, whereas acetone and ethanol had no detrimental effect on folding of the enzyme. BACΔNC was inactive and unstable at extreme pH conditions. Thermal unfolding of the enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl](0.5, N-U), at 1.14 M. BACΔNC was active at the concentrations of urea below 6 M, but it experienced an irreversible unfolding by >8 M denaturant. Taken together, this work lays a foundation for the future structural studies with Bacillus sp. TS-23 α-amylase, a typical member of glycoside hydrolases family 13.
环境变量可以显著影响蛋白质分子的折叠和稳定性。在本研究中，通过戊二醛交联、分析超速离心和各种光谱技术详细研究了截短芽孢杆菌 TS-23 α-淀粉酶（BACΔNC）的生物物理特性。通过交联实验和分析超速离心，我们证明了 BACΔNC 在溶液中的寡聚状态为单体。远紫外圆二色性分析表明，在存在各种金属离子和 SDS 的情况下，BACΔNC 的二级结构发生了显著变化，而丙酮和乙醇对酶的折叠没有不利影响。BACΔNC 在极端 pH 条件下失活且不稳定。发现酶的热变性高度不可逆。天然酶在~0.2 M 盐酸胍（GdnHCl）以上开始展开，并在 1.14 M 时达到展开中间态[GdnHCl](0.5, N-U)。BACΔNC 在低于 6 M 的尿素浓度下具有活性，但在>8 M 变性剂下经历不可逆展开。综上所述，这项工作为未来对属于糖苷水解酶家族 13 的典型成员芽孢杆菌 TS-23 α-淀粉酶的结构研究奠定了基础。
