Chemical modification of carboxylic residues in a cyclodextrin glucanotransferase and its implication in the hydrolysis/transglycosylation ratio of the α-amylase family

The product selectivity varies notably in the enzymes of family 13 of the glycosyl hydrolases, α-amylase family, despite their similar catalytic site (three COOH groups involved) and overall architecture. For example, α-amylases are strongly hydrolytic enzymes, whereas cyclodextrin glycosyltransferases (CGTases) are essentially transglycosylases. Chemical modification of the carboxylic groups (using a water soluble carbodiimide and three different amino nucleophiles) of CGTase from Thermoanaerobacter in absence or presence of a reversible inhibitor has been carried out. In contrast with most hydrolytic enzymes of the α-amylase family, in which carbodiimide modification produces an inactivation effect, the resulting CGTases kept residual activities in the range 22–50% for cyclization and coupling, and 39–80% for disproportionation and hydrolysis. In addition, the selectivity to cyclodextrins and the production of oligosaccharides were not significantly altered when tested under industrial conditions. By amino acid analysis and MALDI-TOF mass spectrometry, it was determined that 4–5 COOH residues were modified. The three carboxylic residues implicated in the active-site (Asp230, Glu258 and Asp329) have a very low water accessibility (<7 Å2). This may help to explain the high transglycosylation/hydrolysis ratio of CGTases in comparison with other α-amylase enzymes.