Limits...
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.


Related in: MedlinePlus

General characteristics of Nox from Lactobacillus pentosus (LpNox).LpNox activity was investigated in regard of (A) pH, (B) buffer type, and (C) temperature. (D) The kinetic parameters were determined by fitting the data of LpNox activity vs. NADH concentration to the Michaelis–Menten equation. (E) No influence of DTT on the thermal stability of LpNox was seen. (F) The half-life of LpNox at its temperature optimum (37°C) was determined by fitting the data points to an exponential decay equation. The error bars indicate the standard deviation of three measurements.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4584968&req=5

Figure 2: General characteristics of Nox from Lactobacillus pentosus (LpNox).LpNox activity was investigated in regard of (A) pH, (B) buffer type, and (C) temperature. (D) The kinetic parameters were determined by fitting the data of LpNox activity vs. NADH concentration to the Michaelis–Menten equation. (E) No influence of DTT on the thermal stability of LpNox was seen. (F) The half-life of LpNox at its temperature optimum (37°C) was determined by fitting the data points to an exponential decay equation. The error bars indicate the standard deviation of three measurements.

Mentions: LpNox was investigated for its suitability as a regeneration enzyme. Therefore, it was tested under various conditions to determine influences on the activity (Figure 2).


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)

General characteristics of Nox from Lactobacillus pentosus (LpNox).LpNox activity was investigated in regard of (A) pH, (B) buffer type, and (C) temperature. (D) The kinetic parameters were determined by fitting the data of LpNox activity vs. NADH concentration to the Michaelis–Menten equation. (E) No influence of DTT on the thermal stability of LpNox was seen. (F) The half-life of LpNox at its temperature optimum (37°C) was determined by fitting the data points to an exponential decay equation. 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 2: General characteristics of Nox from Lactobacillus pentosus (LpNox).LpNox activity was investigated in regard of (A) pH, (B) buffer type, and (C) temperature. (D) The kinetic parameters were determined by fitting the data of LpNox activity vs. NADH concentration to the Michaelis–Menten equation. (E) No influence of DTT on the thermal stability of LpNox was seen. (F) The half-life of LpNox at its temperature optimum (37°C) was determined by fitting the data points to an exponential decay equation. The error bars indicate the standard deviation of three measurements.
Mentions: LpNox was investigated for its suitability as a regeneration enzyme. Therefore, it was tested under various conditions to determine influences on the activity (Figure 2).

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.


Related in: MedlinePlus