Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis.
Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. B1001 catalyzed the production predominantly of alpha-cyclodextrin (CD) from starch (Tachibana, Y. et al., Appl. Environ. Microbiol., 65, 1991-1997, 1999). The CGTase gene (cgtA) from this strain was cloned and sequenced. It was composed of 2217 nucleotides, and encoded a protein (739 amino acids) with a molecular mass of 83,240 Da. Recombinant CgtA expressed in Escherichia coli also catalyzed the production predominantly of alpha-CD from starch, as did native CgtA from strain B1001. Based on a substrate binding model of Bacillus circulans no. 8 CGTase, Tyr100, Trp191 and Tyr267 were specified to locate the spiral amylose and to minimize the size of the CD by saccharide aromatics interaction. In order to determine the critical residue for catalyzing production predominantly of alpha-CD, site-directed mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->Tyr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe). Analysis of the reaction products by HPLC revealed that the mutant enzyme Y267W produced more beta- and gamma-CD than the wild-type enzyme. However, the other mutants still produced high levels of alpha-CD, suggesting that Tyr267 plays a critical role in alpha-CD production catalyzed by B1001 CGTase.
Yamamoto
T
T
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.
Fujiwara
S
S
Tachibana
Y
Y
Takagi
M
M
Fukui
K
K
Imanaka
T
T