Site-saturation mutagenesis of proline 176 in Cyclodextrin Glucosyltransferase from Bacillus sp. Y112 effects product specificity and enzymatic properties

Abstract Based on the analysis of amino acid sequence and simulated structure, saturation mutagenesis was performed to explore the role of the site p176 of cyclodextrin glucosytransferase (CGTase) from Bacillus sp. Y112. Compared to the wild-type, mutant P176 G showed 10.4% improvement in conversion from starch to cyclodextrins (CDs), whose β-CD yield increased by 6% and α-CD yield decreased by 8%. Mutants P176 L and P176I were increased by 7.9% and 9.4% on CDs production, indicating replement of hydrophobic amino acids significantly improved in cyclization activity. Kinetics studies indicated the substrate affinity of P176 G and P176 K were increase by 13% and 14%, and the catalytic efficiency of P176 K was increase by 14%. In addition, the optimal temperature of mutants transformed from 50℃ to 40℃ and the optimal pH shifted from 10.0 to 8.0. These results indicate that the site P176 plays a critial role in catalytic activity, product specificity and enzymatic properties of CGTase.