Altering substrate specificity of alpha-amylase by bioimprinting with beta-cyclodextrin

Abstract

The aim of this study was to alter the substrate specificity of ∞-amylase by the combination of bioimprinting with ®-cyclodextrin (®-CD) and immobilization method. ∞-Amylase was first derivatized with itaconic anhydride to have enough attachment points for enzyme crosslinking. It was determined that the optimum ratio (w/w) of protein to itaconic anhydride was 1:5 which resulted in derivatization degree of 60.7% and the remaining activity of the derivatized enzyme was 93%. The derivatized ∞-amylase was then bioimprinted with ®-CD and immobilized using ethylene glycol dimethacrylate (EGDMA) and 2,2’-azobis (2-methylpropionitrile) (AIBN). It was found that the optimal condition for bioimprinting process was to incubate 30 mg/ml of ∞-amylase with 25 mg/ml of ®-CD in 50 mM sodium acetate buffer pH 6.0 for 30 minutes. And the optimal condition for crosslinking procedure was to use 10 mg/ml of ∞-amylase suspended in cyclohexane with the addition of 4 mg/ml AIBN and EGDMA at the final concentration of 0.82 M under UV irradiation ([gamma] = 240 nm) for 3 hours. The CDase, cyclization and dextrinizing activity of crosslinked imprinted protein (CLIP-∞-amylase) were investigated. The results obtained suggested that the CDase and cyclization activity of CLIP-∞-amylase could not be generated by imprinting with ®-CD under the investigated conditions. Nevertheless, the dextrinizing activity of CLIP-∞-amylase could be altered. The dextrinizing activity was found to be only 42.4% in comparison with that of the crosslinked form where the enzyme was crosslinked without the imprinted molecule.