- 作者: Chii-Ling Jeang ; Chiung-Hua Wung ; Ban-Yang Chang ; Sheng-Shyong Yeh ; Duah-Wern Lour
- 中文摘要: Cyclodextrin glucanotransferase (CGTase, EC 2. 4. 1. 19) converts starch and related .alpha.-1,4-glucans to cyclodextrin (CD). Our previous studies of the enzyme have suggested that E344 on the polypeptide is crucial to the enzyme activity. Mutational analysis of CGTase was performed to confirm this idea. Three mutant CGTases containing either E344D, E344K or E344L substitution were overexpressed in Escherichia coli. However, only the wildtype and E344D CGTases became soluble when expressed at 20 .degree.C. These two enzymes were purified to homogeniety from E. coli cells after .beta.-CD and Ni-NTA affinity chromatographies. The Km values of the authentic Bacillus macerans CGTase (2.10 mM), and of the wild-type (0.58 mM) and E344D (1.05 mM) CGTases purified from E .coli were different. The kcat values of the three CGTases were 99.8, 26.5 and 90.7 s/sup -1/, respectively. The percentage of .alpha.-CD production was 18.4% for the authentic CGTase, 24.9% for the wild-type and 14.5% for the E344D CGTases purified from E. coli. The changes of both the coupling and cyclization activities of CGTase caused by E344D suggest that E344 is important to the catalytic function of CGTase.
- 英文摘要: --
- 中文關鍵字: cyclodextrin glucanotransferase, site-directed mutation, overexpression, CD production
- 英文關鍵字: --