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

Purification of NOXs and characterization of the optimized opt-nox expression levels by SDS-PAGE.(a) 10% SDS-PAGE analysis of the purification opt-nox. M: protein marker; Lane 1: bacterium (harboring pET-32a) induced by IPTG; Lane 2: recombinant bacterium (harboring pET-32a-opt-nox) induced by IPTG; Lane 3: purified opt-nox with His-tag. (b) Expression levels of optimized NOXs in BL21 (DE3) were analyzed by 10% SDS-PAGE. Lane M: molecular mass markers. Lane CK: BL21 (DE3) without nox gene. Lane T01 to T08 are different mutations. Lane opt-nox: the whole optimization of the nox sequence.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128412.g006: Purification of NOXs and characterization of the optimized opt-nox expression levels by SDS-PAGE.(a) 10% SDS-PAGE analysis of the purification opt-nox. M: protein marker; Lane 1: bacterium (harboring pET-32a) induced by IPTG; Lane 2: recombinant bacterium (harboring pET-32a-opt-nox) induced by IPTG; Lane 3: purified opt-nox with His-tag. (b) Expression levels of optimized NOXs in BL21 (DE3) were analyzed by 10% SDS-PAGE. Lane M: molecular mass markers. Lane CK: BL21 (DE3) without nox gene. Lane T01 to T08 are different mutations. Lane opt-nox: the whole optimization of the nox sequence.

Mentions: Opt-nox activities with different inducing time (1, 2, 4, 6, 8 h) were compared in order to determine the optimum inducing time (Fig 5b). NOX activity in cell extract of the opt-nox was 73.3 U/mg after 2 h induction, and it was 2.5-folds of the wild type. Then his-tagged opt-nox was purified by using HisTrap HP column (Table 4) and the specific activity of the purified enzyme was 213.8 U/mg. Purified opt-nox showed a single band in 10% SDS-PAGE (Fig 6a). SDS-PAGE was used to analyze the quantity of NOX in order to exhibit the improvement of the protein expression level (Fig 6b), which demonstrated that protein expression amount and specific activity presented certain positive correlation.


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)

Purification of NOXs and characterization of the optimized opt-nox expression levels by SDS-PAGE.(a) 10% SDS-PAGE analysis of the purification opt-nox. M: protein marker; Lane 1: bacterium (harboring pET-32a) induced by IPTG; Lane 2: recombinant bacterium (harboring pET-32a-opt-nox) induced by IPTG; Lane 3: purified opt-nox with His-tag. (b) Expression levels of optimized NOXs in BL21 (DE3) were analyzed by 10% SDS-PAGE. Lane M: molecular mass markers. Lane CK: BL21 (DE3) without nox gene. Lane T01 to T08 are different mutations. Lane opt-nox: the whole optimization of the nox sequence.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128412.g006: Purification of NOXs and characterization of the optimized opt-nox expression levels by SDS-PAGE.(a) 10% SDS-PAGE analysis of the purification opt-nox. M: protein marker; Lane 1: bacterium (harboring pET-32a) induced by IPTG; Lane 2: recombinant bacterium (harboring pET-32a-opt-nox) induced by IPTG; Lane 3: purified opt-nox with His-tag. (b) Expression levels of optimized NOXs in BL21 (DE3) were analyzed by 10% SDS-PAGE. Lane M: molecular mass markers. Lane CK: BL21 (DE3) without nox gene. Lane T01 to T08 are different mutations. Lane opt-nox: the whole optimization of the nox sequence.
Mentions: Opt-nox activities with different inducing time (1, 2, 4, 6, 8 h) were compared in order to determine the optimum inducing time (Fig 5b). NOX activity in cell extract of the opt-nox was 73.3 U/mg after 2 h induction, and it was 2.5-folds of the wild type. Then his-tagged opt-nox was purified by using HisTrap HP column (Table 4) and the specific activity of the purified enzyme was 213.8 U/mg. Purified opt-nox showed a single band in 10% SDS-PAGE (Fig 6a). SDS-PAGE was used to analyze the quantity of NOX in order to exhibit the improvement of the protein expression level (Fig 6b), which demonstrated that protein expression amount and specific activity presented certain positive correlation.

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