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Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration.
- Source :
- PLoS ONE, Vol 10, Iss 6, p e0128412 (2015)
- Publication Year :
- 2015
- Publisher :
- Public Library of Science (PLoS), 2015.
-
Abstract
- NADH oxidases (NOXs) play an important role in maintaining balance of NAD+/NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coli BL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as Vmax(Glycerol) 20 μM/min, Km(Glycerol) 19.4 mM, Vmax (NADH) 12.5 μM/min and Km (NADH) 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis.
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 10
- Issue :
- 6
- Database :
- Directory of Open Access Journals
- Journal :
- PLoS ONE
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.2803ab1dae8246ce8d3ff2c3f9dcc4eb
- Document Type :
- article
- Full Text :
- https://doi.org/10.1371/journal.pone.0128412