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Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis. | LitMetric

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis.

J Biotechnol

Instituto de Tecnologia Química e Biológica António Xavier, Universidade NOVA de Lisboa, Av. da República, Oeiras 2780-157, Portugal. Electronic address:

Published: August 2024

AI Article Synopsis

  • Protein engineering is important for improving enzymes in industrial biocatalysis, specifically focusing on a bacterial dioxygenase called NOV1 that converts isoeugenol into vanillin, a widely-used flavoring agent.
  • The study utilized a tool called Zymspot to identify beneficial distant mutations and created 41 enzyme variants, leading to two promising versions that showed a nearly 10-fold increase in activity and up to 40-fold improved stability compared to the wild-type enzyme.
  • The optimized enzyme variant 1D2 demonstrated effective bioconversion, producing significantly more vanillin than the wild-type, highlighting the potential of distal protein engineering for enhancing enzyme properties and its relevance for biotechnological applications.

Article Abstract

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Å from the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

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Source
http://dx.doi.org/10.1016/j.jbiotec.2024.06.012DOI Listing

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