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Expression and characterization of l-arabinose isomerase from Geobacillus stearothermophilus for improved activity under acidic condition.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2020 Nov; Vol. 175, pp. 105692. Date of Electronic Publication: 2020 Jul 16. - Publication Year :
- 2020
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Abstract
- A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction. Previously, we obtained a mutant of l-AI, H18T, from Geobacillus stearothermophilus with greater substrate specificity. Although H18T possessed excellent thermostability, its activity under acidic conditions was not optimal. Here, we successfully obtained a potential variant of the H18T protein, H18T-Y234C, which achieved improved activity at pH 6.0, based on random mutagenesis using error-prone PCR around the binding pocket area of H18T. This double H18T-Y234C mutant possessed 1.8-fold and 3-fold higher activity at pH 6.0 than the parent H18T and the wild type, thereby broadening the optimal pH range to 6.0-8.0. Mutation from Tyr to Cys at residue 234 had little effect on the secondary structure of L-AI. Furthermore, the formation of disulfide bonds was not detected. Thus, the improvement of activity at pH 6.0 is probably caused by the change in the binding pocket area involving residue 234. This study offers insight into the importance of residue 234 in improving the activity under acidic conditions.<br /> (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Subjects :
- Enzyme Stability
Geobacillus stearothermophilus enzymology
Hot Temperature
Hydrogen-Ion Concentration
Recombinant Proteins biosynthesis
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Aldose-Ketose Isomerases biosynthesis
Aldose-Ketose Isomerases chemistry
Aldose-Ketose Isomerases genetics
Aldose-Ketose Isomerases isolation & purification
Bacterial Proteins biosynthesis
Bacterial Proteins chemistry
Bacterial Proteins genetics
Bacterial Proteins isolation & purification
Gene Expression
Geobacillus stearothermophilus genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 175
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 32681957
- Full Text :
- https://doi.org/10.1016/j.pep.2020.105692