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

Optimal induce time of the T01-08, opt-nox and the wild type.(a)Comparison of the specific activity of the T01-08 and the wild type with different induced time (1–8h). (b) Optimal inducing time of the opt-nox.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128412.g005: Optimal induce time of the T01-08, opt-nox and the wild type.(a)Comparison of the specific activity of the T01-08 and the wild type with different induced time (1–8h). (b) Optimal inducing time of the opt-nox.

Mentions: Optimum inducing time of the mutant strains T01-08 was determined by comparison of T01-08 activities with different inducing time (1, 2, 4, 6, 8h). It could be noted that the T01-08 exhibited the highest activities after 4 h induction (Fig 5a), which was showed in Table 3. Increasing AT content in mutation area, the specific activity improved. NOX activity in cell extract of the mutants was 59.9 U/mg, which was 2.0-folds of the wild type (28.9 U/mg).


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)

Optimal induce time of the T01-08, opt-nox and the wild type.(a)Comparison of the specific activity of the T01-08 and the wild type with different induced time (1–8h). (b) Optimal inducing time of the opt-nox.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128412.g005: Optimal induce time of the T01-08, opt-nox and the wild type.(a)Comparison of the specific activity of the T01-08 and the wild type with different induced time (1–8h). (b) Optimal inducing time of the opt-nox.
Mentions: Optimum inducing time of the mutant strains T01-08 was determined by comparison of T01-08 activities with different inducing time (1, 2, 4, 6, 8h). It could be noted that the T01-08 exhibited the highest activities after 4 h induction (Fig 5a), which was showed in Table 3. Increasing AT content in mutation area, the specific activity improved. NOX activity in cell extract of the mutants was 59.9 U/mg, which was 2.0-folds of the wild type (28.9 U/mg).

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