Yamamoto T, Shiraki K, Fujiwara S, Takagi M, Fukui K, Imanaka T
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, 565-0871, Japan.
Biochem Biophys Res Commun. 1999 Nov;265(1):57-61. doi: 10.1006/bbrc.1999.1629.
The in vitro heat effect on protein characteristics of thermostable enzyme was examined using a cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from the hyperthermophilic archaeon Thermococcus sp. B1001 as a model protein. The recombinant form of CGTase was obtained as an inclusion body from Escherichia coli cells harboring a plasmid which carried the B1001 CGTase gene (cgtA). CGTase was solubilized by 6 M urea, refolded, purified to homogeneity, and heat treated at 80 degrees C for 20 min. Enzyme characteristics were examined compared with those of unheated CGTase. Cyclization activity was increased by in vitro heat treatment, while hydrolysis activity was decreased. The heated and unheated CGTases were analyzed for structures by circular dichroism (CD). The near- and far-UV CD spectra indicated that the structure of unheated CGTase with low cyclization activity was different from that of heated CGTase with high activity. Differential scanning calorimetry of unheated CGTase showed two absorption peaks at 87 and 106 degrees C with increasing temperature. After heat treatment, the minor peak at 87 degrees C disappeared, suggesting that heat-dependent structural conversion occurred in CGTase. These results indicate that the thermal environment plays an important role for the protein folding process of thermostable CGTase.
以嗜热古菌嗜热栖热菌（Thermococcus sp.）B1001来源的环糊精葡聚糖转移酶（CGTase，EC 2.4.1.19）作为模型蛋白，研究了体外加热对热稳定酶蛋白特性的影响。携带B1001 CGTase基因（cgtA）的质粒转入大肠杆菌细胞，重组CGTase以包涵体形式获得。用6 M尿素溶解CGTase，复性，纯化至均一，然后在80℃热处理20分钟。将酶的特性与未加热的CGTase进行比较。体外热处理使环化活性增加，而水解活性降低。通过圆二色性（CD）分析加热和未加热的CGTase的结构。近紫外和远紫外CD光谱表明，环化活性低的未加热CGTase的结构与活性高的加热CGTase的结构不同。未加热的CGTase的差示扫描量热法显示，随着温度升高，在87℃和106℃出现两个吸收峰。热处理后，87℃的小峰消失，表明CGTase发生了热依赖性结构转变。这些结果表明，热环境对热稳定CGTase的蛋白质折叠过程起着重要作用。
