In vitro heat effect on functional and conformational changes of cyclodextrin glucanotransferase from hyperthermophilic archaea.
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.
Yamamoto
T
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka, Suita, 565-0871, Japan.
Shiraki
K
K
Fujiwara
S
S
Takagi
M
M
Fukui
K
K
Imanaka
T
T
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D019712:Thermococcus / Q000201:enzymology*
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase