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Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration.

Fang B, Jiang W, Zhou Q, Wang S - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China; The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian, 361005, China.

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.

No MeSH data available.


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Bioconversion of glycerol by fused glycerol dehydrogenase and NADH oxidase coupled with NADH regeneration.
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pone.0128412.g001: Bioconversion of glycerol by fused glycerol dehydrogenase and NADH oxidase coupled with NADH regeneration.

Mentions: Glycerol dehydrogenases (GDHs) play crucial roles in the pathway of glycerol metabolism for the production of dioxyacetone (DHA) and 1, 3-propanediol (1, 3-PD). GDHs are also widely used in medical diagnosis and glycerol concentration analysis in fermentation process [22, 23]. Coupling with NADH oxidases, glycerol dehydrogenase catalyzes glycerol to dioxyacetone with NAD+ regeneration, which overcome the disadvantage of expensive consumption of NAD+ (Fig 1). Based on end-to-end fusion technique, many fusion enzymes have been developed [24, 25], which obtained enhanced catalysis efficiency.


Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration.

Fang B, Jiang W, Zhou Q, Wang S - PLoS ONE (2015)

Bioconversion of glycerol by fused glycerol dehydrogenase and NADH oxidase coupled with NADH regeneration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482596&req=5

pone.0128412.g001: Bioconversion of glycerol by fused glycerol dehydrogenase and NADH oxidase coupled with NADH regeneration.
Mentions: Glycerol dehydrogenases (GDHs) play crucial roles in the pathway of glycerol metabolism for the production of dioxyacetone (DHA) and 1, 3-propanediol (1, 3-PD). GDHs are also widely used in medical diagnosis and glycerol concentration analysis in fermentation process [22, 23]. Coupling with NADH oxidases, glycerol dehydrogenase catalyzes glycerol to dioxyacetone with NAD+ regeneration, which overcome the disadvantage of expensive consumption of NAD+ (Fig 1). Based on end-to-end fusion technique, many fusion enzymes have been developed [24, 25], which obtained enhanced catalysis efficiency.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China; The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, 361005, China; The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian, 361005, China.

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.

No MeSH data available.


Related in: MedlinePlus