Engineering a chimeric acid-stable α-amylase-glucoamylase (Amy-Glu) for one step starch saccharification

Abstract

For saccharifying starch in one step, a chimeric biocatalyst (Amy-Glu) was generated from engineered α-amylase (Ba-Gt-amy) of Bacillus acidicola and glucoamylase (Glu) gene of Aspergillus niger. In order to join two enzymes, a linker peptide of 25 amino acids was used. Chimeric Amy-Glu was expressed in E. coli. Glu is of 75kDa, while Amy-Glu is of 145kDa. Both Amy-Glu and Glu displayed similar pH profile with good activity in the acidic pH range like that of Ba-Gt-amy with optimum at pH 4.0. All three enzymes (Ba-Gt-amy, Amy-Glu and glucoamylase) exhibited activity in the temperature range between 40 and 70°C with optimum at 60°C. Amy-Glu and Glu have T1/2 of 90 and 70min at 60 and 70°C, respectively. The Km, Vmax and Kcat values of Glu (soluble starch) are 0.34mgmL−1, 606μmolmg−1min−1 and 727s−1, while for Amy-Glu are 0.84mgmL−1, 13,886μmolmg−1min−1 and 4.2×104s−1, respectively. The end product analysis suggested that Amy-Glu retains the activity of both parental enzymes and forms maltodextrins along with glucose as the major products. Amy-Glu saccharifies wheat and corn starches more efficiently than the Ba-Gt-amy and glucoamylase.