1. Engineering a chimeric acid-stable α-amylase-glucoamylase (Amy-Glu) for one step starch saccharification.
- Author
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Parashar D and Satyanarayana T
- Subjects
- Aspergillus niger enzymology, Bacillus enzymology, Biocatalysis, Cloning, Molecular, Enzyme Stability, Escherichia coli genetics, Glucan 1,4-alpha-Glucosidase metabolism, Hydrolysis, Recombinant Fusion Proteins metabolism, Glucan 1,4-alpha-Glucosidase chemistry, Glucan 1,4-alpha-Glucosidase genetics, Protein Engineering, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Starch metabolism, alpha-Amylases genetics
- 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 T
1/2 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-1 min-1 and 727s-1 , while for Amy-Glu are 0.84mgmL-1 , 13,886μmolmg-1 min-1 and 4.2×104 s-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., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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