Limits...
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.


Related in: MedlinePlus

Determination of kinetic parameters of GDH-NOX.(a) Glycerol; (b) NADH. Experiment condition: glycerol concentration (0.01, 0.125, 0.014, 0.025, 0.05M), NADH concentration (20, 40, 60, 100, 200 μM). pH 7.0, 37°C.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482596&req=5

pone.0128412.g009: Determination of kinetic parameters of GDH-NOX.(a) Glycerol; (b) NADH. Experiment condition: glycerol concentration (0.01, 0.125, 0.014, 0.025, 0.05M), NADH concentration (20, 40, 60, 100, 200 μM). pH 7.0, 37°C.

Mentions: The full-length sequence of the fusion gdh-nox gene was obtained. The enzymes were purified by using HisTrap HP column and SDS—PAGE showed a band of the purified GDH-NOX with His-tag (Fig 7). Among the bifunctional fused enzyme, the optimal pH for NOX was pH 7.0 while for the GDH was 11.0 (Fig 8a and 8b). The optimum temperature of the GDH and NOX among the GDH-NOX was 45°C and 37°C, respectively (Fig 8c and 8d). The specific activities of the GDH and NOX of the GDH-NOX were 15.1 U/mg and 15.7 U/mg, respectively. Kinetic parameters of the GDH-NOX for two substrates, 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 (Fig 9).


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)

Determination of kinetic parameters of GDH-NOX.(a) Glycerol; (b) NADH. Experiment condition: glycerol concentration (0.01, 0.125, 0.014, 0.025, 0.05M), NADH concentration (20, 40, 60, 100, 200 μM). pH 7.0, 37°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128412.g009: Determination of kinetic parameters of GDH-NOX.(a) Glycerol; (b) NADH. Experiment condition: glycerol concentration (0.01, 0.125, 0.014, 0.025, 0.05M), NADH concentration (20, 40, 60, 100, 200 μM). pH 7.0, 37°C.
Mentions: The full-length sequence of the fusion gdh-nox gene was obtained. The enzymes were purified by using HisTrap HP column and SDS—PAGE showed a band of the purified GDH-NOX with His-tag (Fig 7). Among the bifunctional fused enzyme, the optimal pH for NOX was pH 7.0 while for the GDH was 11.0 (Fig 8a and 8b). The optimum temperature of the GDH and NOX among the GDH-NOX was 45°C and 37°C, respectively (Fig 8c and 8d). The specific activities of the GDH and NOX of the GDH-NOX were 15.1 U/mg and 15.7 U/mg, respectively. Kinetic parameters of the GDH-NOX for two substrates, 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 (Fig 9).

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