Cyclodextrin Glucanotransferase from Bacillus circulans DF 9R: product specificity at the -6 subsite

COSTA HERNÁN; HIDALGO AURELIO; FERRAROTTI SUSANA; BERENGUER JOSÉ; BISCOGLIO DE JIMÉNEZ BONINO MIRTHA

Tucuman, Argentina

Congreso; SAIB 2009; 2009

Sociedad Argentina de Investigación Bioquímica

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is a member of the GH13 family that catalyses the conversion of starch into cyclodextrins which are important for industrial applications. Nine substrate binding subsites have been described in CGTases. Interactions at the -6 subsite (Tyr167, Gly179, Gly180, Asn193, Asp196) activate the enzyme via an induced-fit mechanism. All the CGTases whose primary structure is already known have Gly at position 179 and it has been suggested that the absence of side chains in this residue is a requirement for substrate binding favoring such mechanism. Nevertheless, we have demonstrated that CGTase from Bacillus circulans DF 9R is the only one posessing Gln instead of Gly in that position. The gen of this CGTase was cloned into pET22b(+) plasmid and two mutants were obtained: Gln179Gly and Gln179Leu. The corresponding recombinant proteins were expressed in E. coli BL21 and purified by affinity-cromatography. The Gln179Gly mutant showed higher hidrolytic activity than both the wild type recombinant and the Gln179Leu mutant enzymes. This work is a contribution to the knowledge of CGTase structure-function relationship, particularly about product specificity.