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

Baishan Fang; Wei Jiang; Qiang Zhou; Shizhen Wang

doi:10.1371/journal.pone.0128412

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

PLoS One. 2015 Jun 26;10(6):e0128412. doi: 10.1371/journal.pone.0128412. eCollection 2015.

Authors

Baishan Fang¹, Wei Jiang², Qiang Zhou², Shizhen Wang²

Affiliations

¹ 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.
² 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular
Codon*
Coenzymes
Enzyme Activation
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression*
Gene Fusion*
Glycerol / metabolism
Multienzyme Complexes / genetics*
Multienzyme Complexes / metabolism*
NADH, NADPH Oxidoreductases / genetics*
NADH, NADPH Oxidoreductases / metabolism*
Protein Engineering
Recombinant Fusion Proteins* / genetics
Recombinant Fusion Proteins* / isolation & purification
Regeneration
Sugar Alcohol Dehydrogenases / genetics*
Sugar Alcohol Dehydrogenases / metabolism*

Substances

Codon
Coenzymes
Multienzyme Complexes
Recombinant Fusion Proteins
Sugar Alcohol Dehydrogenases
glycerol dehydrogenase
NADH oxidase
NADH, NADPH Oxidoreductases
Glycerol

Grants and funding

This work was supported by the National Natural Science Foundation of China (No. 41176111, No. 41306124), the State Key Program of the National Natural Science Foundation of China (No. 21336009), and the Fundamental Research Funds for the Central Universities (No. 2013121029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.