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A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

Nowak C, Beer B, Pick A, Roth T, Lommes P, Sieber V - Front Microbiol (2015)

Bottom Line: We found that the heterologously overexpressed enzyme only contained 13% FAD.In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics.The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors.

View Article: PubMed Central - PubMed

Affiliation: Chair of Chemistry of Biogenic Resources, Straubing Centre of Science, Department Life Science Engineering, Technische Universität München, Straubing Germany.

ABSTRACT
The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

No MeSH data available.


Kinetic measurements of LpNox with biomimetic cofactors. The kinetic parameters were determined by fitting the data of LpNox activity vs. cofactor concentration to the Michaelis–Menten equation (A, MNAH, B, BNAH). The error bars indicate the standard deviation of three measurements.
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Figure 3: Kinetic measurements of LpNox with biomimetic cofactors. The kinetic parameters were determined by fitting the data of LpNox activity vs. cofactor concentration to the Michaelis–Menten equation (A, MNAH, B, BNAH). The error bars indicate the standard deviation of three measurements.

Mentions: For the determination of the kinetic constants of LpNox with the biomimetic cofactors a fluorescence assay was used, because high concentrations of reduced cofactors exceed the maximal absorbance for photometric measurements. Both could be fitted to the Michaelis-Menten equation (Figure 3). Km and vmax (kcat) of both cofactors are in the same range with BNAH showing a slightly lower Km and higher vmax than MNAH. Therefore, kcat/Km for BNAH is 1.4 higher than for MNAH (Table 3).


A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

Nowak C, Beer B, Pick A, Roth T, Lommes P, Sieber V - Front Microbiol (2015)

Kinetic measurements of LpNox with biomimetic cofactors. The kinetic parameters were determined by fitting the data of LpNox activity vs. cofactor concentration to the Michaelis–Menten equation (A, MNAH, B, BNAH). The error bars indicate the standard deviation of three measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4584968&req=5

Figure 3: Kinetic measurements of LpNox with biomimetic cofactors. The kinetic parameters were determined by fitting the data of LpNox activity vs. cofactor concentration to the Michaelis–Menten equation (A, MNAH, B, BNAH). The error bars indicate the standard deviation of three measurements.
Mentions: For the determination of the kinetic constants of LpNox with the biomimetic cofactors a fluorescence assay was used, because high concentrations of reduced cofactors exceed the maximal absorbance for photometric measurements. Both could be fitted to the Michaelis-Menten equation (Figure 3). Km and vmax (kcat) of both cofactors are in the same range with BNAH showing a slightly lower Km and higher vmax than MNAH. Therefore, kcat/Km for BNAH is 1.4 higher than for MNAH (Table 3).

Bottom Line: We found that the heterologously overexpressed enzyme only contained 13% FAD.In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics.The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors.

View Article: PubMed Central - PubMed

Affiliation: Chair of Chemistry of Biogenic Resources, Straubing Centre of Science, Department Life Science Engineering, Technische Universität München, Straubing Germany.

ABSTRACT
The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

No MeSH data available.