Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp B1001 by site-directed mutagenesis

Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeo n Thermococcus sp. B1001 catalyzed the production predominantly of a-cyclod extrin (CD) from starch (Tachibana, Y. et al,, Appl. Environ, Microbiol., 6 5, 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 Ha. Recombinant CgtA expresse d in Escherichia colt 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 th e CD by saccharide aromatics interaction. In order to determine the critica l residue for catalyzing production predominantly of alpha-CD, site-directe d mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->T yr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe), Analysi s of the reaction products by HPLC revealed that the mutant enzyme Y267W pr oduced more beta- and gamma-CD than the wild-type enzyme. However, the othe r mutants still produced high levels of alpha-CD, suggesting that Tyr267 pl ays a critical role in alpha-CD production catalyzed by B1001 CGTase.