Recombinant cyclodextrin glycosyltransferase suitable for employment in food industry

CASTILLO DE LAS MERCEDES, JULIETA; RODRIGUEZ GASTÓN, JORGELINA ANDREA; MATIOLI, GRACIETTE; CAMINATA LANDRIEL, SOLEDAD; CANTERO, MARÍA JOSÉ; FENELON, VANDERSON C.; GREGOLIN GIMENEZ, GABRIELA; TABOGA, OSCAR ALBERTO; COSTA, HERNÁN; FERRAROTTI, SUSANA ALICIA

Ciudad Autónima de Buenos Aires

Congreso; Reunión Conjunta Sociedades de Biociencias; 2017

Theenzyme cyclodextrin glycosyltransferase (CGTase) biotransforms the starch intocyclic oligosaccharides with different degrees of polymerization calledcyclodextrins (CD), widely used in different industries. These compounds have ahydrophobic internal cavity and a hydrophilic surface, whereby they can forminclusion complexes with many molecules and thus modify their physical andchemical properties. Escherichia coli is the main productionsystem for recombinant proteins. However, downstream processing is laborious. In addition, E. coli cell envelope containslipopolysaccharides that act as pyrogenic endotoxins in humans and othermammals thus preventing the use of this bacterium in the obtention of productsto the pharmaceutical and food industries. Given the industrial utility of theCGTase and its main products, the objective of this work is the production of CGTase isolatedfrom Bacillus circulans in Pichia pastoris, a GRAS (generallyrecognized as safe) system. The CGTase gene was amplifiedby PCR and cloned into the expression vector pPICZαA, with which P. pastoris cells were transformed. Expression was performed by the addition of methanolto the culture medium and the presence of the enzyme in aliquots of the culturesupernatant, collected at different post-induction times, was analyzed bySDS-PAGE and zymogram. The recombinant enzyme was purified by affinity to α-CD and its cyclization activity was determined. In this way, a recombinant CGTase was producedin the culture medium, which the subsequent one-step purification allows tohave homogeneous preparations with high activity, suitable for use in the foodindustry.