Improved activity of β-cyclodextrin glycosyltransferase from Bacillus sp. N-227 via mutagenesis of the conserved residues.

β-Cyclodextrin glycosyltransferase (β-CGTase) belongs to the α-amylase family of enzymes and converts starch to cyclic oligosaccharides called β-cyclodextrins (β-CD). The β-CGTase from alkalophilic Bacillus sp. N-227 was separately mutagenized to give three site-directed β-CGTase mutants, Y127F, R254F and D355R, that showed enhanced cyclization activity towards a starch substrate from 1.64 to 2.1-folds. Kinetic studies indicate that the mutants had higher affinity towards the substrate than the wild type β-CGTase. The Y127F mutant had the highest affinity which was indicated by the lowest K m of 15.30 mM and the highest catalytic activity. Increasing hydrophobicity around the catalytic center appeared to favor the cyclization activity of the mutants. The β-CGTase and the three mutants showed optimal enzyme activity at 60 °C and pH 6.0. All the enzymes were stable for at least 60 min across a wide pH range (5.0-7.0).