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

• The residue Pro-176 located in the active groove of the CGTase was replaced by other 15 amino acids residues. • The mutant P176G is important for the β-CD specificity. • The cyclization activity of mutants P176I and P176L both improve. • The Pro at position 176 play an important role in enzymatic properties. 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 P176G 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 P176L and P176I were increased by 7.9 % and 9.4 % on CDs production, indicating replacement of hydrophobic amino acids significantly improved in cyclization activity. Kinetics studies indicated the substrate affinity of P176G and P176K were increase by 13 % and 14 %, and the catalytic efficiency of P176K 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 critical role in catalytic activity, product specificity and enzymatic properties of CGTase. [ABSTRACT FROM AUTHOR]