title: 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 T1/2of 90 and 70min at 60 and 70°C, respectively. The Km, Vmaxand Kcatvalues of Glu (soluble starch) are 0.34mgmL-1, 606μmolmg-1min-1and 727s-1, while for Amy-Glu are 0.84mgmL-1, 13,886μmolmg-1min-1and 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.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Parashar
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address: tsnarayana@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D013213:Starch; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase
keywords: Chimera; Glucoamylase; Raw starch; Saccharification; α-Amylase