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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.


Related in: MedlinePlus

Alignment of wild type nucleotide sequence (nox) with the optimized sequence (Nox-opt).Identical residues were highlighted in black background.
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pone.0128412.g004: Alignment of wild type nucleotide sequence (nox) with the optimized sequence (Nox-opt).Identical residues were highlighted in black background.

Mentions: The first optimization strategy was based on improving the AT content of 2–6 codons downstream of the initiation codon. The mutants (T01-08) were obtained by site directed mutagenesis and expressed in BL21 (DE3). Comparison of mutation sites with the wild-type was showed in Table 3. For the second optimization strategy, gene sequence was rearranged for keep the codon usage frequency consistent with the E. coli BL21 (DE3) codon usage frequency. The optimized NOX gene (Nox-opt) was synthesized according to the codon optimization scheme and its sequence comparison with wild-type sequence was showed in Fig 4. Codon adaptation index (CAI) of the optimized sequence of the Nox-opt was 0.53, which was calculated by Codon-Adaptation Tool [33], was higher than that of the wild type (0.39), suggesting the optimized Nox-opt would be better in the expression in the host cell.


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)

Alignment of wild type nucleotide sequence (nox) with the optimized sequence (Nox-opt).Identical residues were highlighted in black background.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128412.g004: Alignment of wild type nucleotide sequence (nox) with the optimized sequence (Nox-opt).Identical residues were highlighted in black background.
Mentions: The first optimization strategy was based on improving the AT content of 2–6 codons downstream of the initiation codon. The mutants (T01-08) were obtained by site directed mutagenesis and expressed in BL21 (DE3). Comparison of mutation sites with the wild-type was showed in Table 3. For the second optimization strategy, gene sequence was rearranged for keep the codon usage frequency consistent with the E. coli BL21 (DE3) codon usage frequency. The optimized NOX gene (Nox-opt) was synthesized according to the codon optimization scheme and its sequence comparison with wild-type sequence was showed in Fig 4. Codon adaptation index (CAI) of the optimized sequence of the Nox-opt was 0.53, which was calculated by Codon-Adaptation Tool [33], was higher than that of the wild type (0.39), suggesting the optimized Nox-opt would be better in the expression in the host cell.

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