Characterization of the Bacillus macerans cyclodextrin glucanotransferase overexpressed in Escherichia coli.
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 wild-type and E344D CGTases became soluble when expressed at 20 degrees 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-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.
Jeang
C L
CL
Department of Food Science, National Chung Hsing University, Taichung, Taiwan, R.O.C.
Wung
C H
CH
Chang
B Y
BY
Yeh
S S
SS
Lour
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase