Enzymatic decontamination of Deoxynivalenol

ENZYMATIC DECONTAMINATION OF DEOXYNIVALENOL

Valeria Della Gala1, Jog Raj2, Hunor Farkaš22, Marko Vasiljević2 and Ditte Hededam Welner1 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark 1

PATENT CO, DOO., Mišićevo, Serbia

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ENZYNOV’2 Conference on Enzymatic Biocatalysis For Industry - Unleashing the power of Enzymes and Biocatalysis for industrial applications. Enzyme production challenges, Sustainability, innovations, and industrial applications. October 26-27, 2023. Biocitech, Paris-Romainville, France.

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BACKGROUND Deoxynivalenol (DON), a mycotoxin produced by the fungus Fusarium graminearum, frequently contaminates crops, negatively impacting plant growth and crop yields.

Its presence in food and feed poses a severe threat to human and animal health1.

DON’s toxicity is related to the hydroxyl group at the C-3 atom, which can exist in two configurations: S-configuration (DON) R-configuration (3-epi-DON)

To date, the crystal structure of one DH (DmDepA) and one AKR

Studies have shown that 3-epi -DON is at

(RlDepB) has been elucidated and,

least 50 times less toxic than DON2.

although various enzymatic activities have been identified, their actual

The conversion of DON into 3-epi -DON,

industrial uses are limited due to lack

which involves the formation of 3-keto -DON,

of understanding and stability issues.

is catalyzed by two classes of enzymes3: PQQ-dependent dehydrogenases (DHs) NADPH-dependent aldo-keto reductases (AKRs)

Aldo-keto reductase

Dehydrogenase

Deoxynivalenol (DON)

3-keto-DON

3-epi-DON

Reduced toxicity

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OBJECTIVE The aim of this study was to identify and develop two robust and efficient biocatalysts with the required industrial characteristics to deactivate DON in animal feed.

MATERIALS & METHODS The study involved a three-step procedure: 1. Discovery of novel PQQ-dependent DHs and NADPH-dependent AKRs. 2. Cloning, expression, and purification of the newly identified DHs and AKRs. 3. Enzymatic characterization, reaction optimization, and engineering.

1. Discovery of novel PQQ-dependent DHs and NADPH-dependent AKRs Amino acid sequence of DmDepA (DH) and RIDepB (AKR)

In silico 3D structure generation and visual inspection Sequence similarity search

Multiple sequence alignment

Candidate homologs

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2. Cloning, expression and purification of the newly identified DHs and AKRs

Codon optimization and plasmid design

Bacterial transformation

Protein expression

HisTrapTM purification

Purified DHs and AKRs

3. Enzymatic characterization, reaction optimization and engineering Load reduction Broad pH and temperatura range for activity Reaction optimization and enzyme engineering Enzymatic assay of DHs and AKRs with DON and 3-keto-DON

HPLC analysis

Thermostability

Biochemical, kinetic and stability characterization

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RESULTS & CONCLUSIONS As a result of this study, it was concluded that: DON epimerization can be a solution for food and feed decontamination. However, reaction conditions need to be optimized.

This procedure allowed for the identification of 13 novel sequences (DHs and AKRs).

Analytical separation of epimers (DON and 3-epi-DON) can be challenging.

Considering these results, in the future, it will be important to determine if: The identified DHs and AKRs enzymes operate optimally under the required industrial conditions.

It is possible to improve stability with rational or semi-rational engineering.

References Kamle, M., et al. Deoxynivalenol: An Overview on Occurrence, Chemistry, Biosynthesis, Health Effects and Its Detection, Management, and Control Strategies in Food and Feed. Microbiol. Res. 13, 292-314 (2022).

2 He, J. W., et al. Toxicology of 3-epi-deoxynivalenol, a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8, Food and Chemical Toxicology, 84, 250-259 (2015).

3 Hassan, Y.I., et al. The enzymatic epimerization of deoxynivalenol by Devosia mutans proceeds through the formation of 3-keto-DON intermediate. Sci Rep, 7, 6929 (2017).

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