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Ammar Al-Shameri1, Dominik L Siebert1, Samuel Sutiono1

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A novel hydrogenase-based system for nicotinamide adenine dinucleotide (NAD+) regeneration outperforms the traditional NADH oxidase (NOX) system. This sustainable biocatalysis method offers improved efficiency and technical simplicity for chemical production.

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Area of Science:

  • Biocatalysis and sustainable chemistry
  • Cofactor regeneration systems
  • Enzyme engineering

Background:

  • Biocatalysis offers a clean route for chemical synthesis, often requiring nicotinamide adenine dinucleotide (NAD+) regeneration.
  • NADH oxidase (NOX) is commonly used for NAD+ regeneration but faces challenges with oxygen supply and mass transfer.
  • Efficient NAD+ regeneration is crucial for the economic viability of biocatalytic processes.

Purpose of the Study:

  • To introduce and evaluate a novel NAD+ regeneration system based on hydrogen evolution using hydrogenase.
  • To compare the performance of the hydrogenase-based system against the traditional NOX system.
  • To demonstrate the applicability of the hydrogenase system in a multi-enzymatic cascade for ketoacid production.

Main Methods:

  • In vitro NAD+ regeneration using hydrogenase and H2 evolution.
  • Integration of the hydrogenase system into a multi-enzymatic cascade for converting sugars to ketoacids.
  • Comparative analysis of NAD+ recycling efficiency and scalability against the NOX system.

Main Results:

  • The hydrogenase-based system achieved significantly higher NAD+ recycling (up to 44,000 mol per mol enzyme) compared to NOX.
  • The system demonstrated superior technical simplicity, flexibility, and overall output.
  • The hydrogenase system effectively produced ketoacids from sugars, with only green H2 as a byproduct, even in the presence of oxygen.

Conclusions:

  • Hydrogenase-based NAD+ regeneration is a scalable and superior alternative to NOX for biocatalysis.
  • This system offers enhanced efficiency, technical advantages, and environmental benefits (green H2 byproduct).
  • The developed system holds significant promise for sustainable chemical production platforms.