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LUND UNIVERSITY LIBRARIES

Characterization of Coniferyl Aldehyde Dehydrogenase and cofactor regeneration with NADH oxidase in the biotransformation from FFCA to FDCA

Li, Chengsi LU (2024) KBTM01 20241
Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
Abstract
This thesis focuses on the characterization of Coniferyl aldehyde dehydrogenase (CALDH) and cofactor regeneration involved in the biotransformation process from 5-Formyl-2-furancarboxylic Acid (FFCA) to 2,5-furandicarboxylic acid (FDCA) using NADH oxidases. FDCA is a building block of bio-based polymers like polyethylene furanoate (PEF), which serves as a sustainable alternative to traditional Polyethylene terephthalate (PET) plastics. However, the biocatalytic process faces efficiency challenges due to, for example, the limited availability of cofactors like NAD+, which are essential for the catalytic activity of enzymes involved in this conversion. In this study, the enzyme CALDH (previously characterized from Gluconobacter oxydans... (More)
This thesis focuses on the characterization of Coniferyl aldehyde dehydrogenase (CALDH) and cofactor regeneration involved in the biotransformation process from 5-Formyl-2-furancarboxylic Acid (FFCA) to 2,5-furandicarboxylic acid (FDCA) using NADH oxidases. FDCA is a building block of bio-based polymers like polyethylene furanoate (PEF), which serves as a sustainable alternative to traditional Polyethylene terephthalate (PET) plastics. However, the biocatalytic process faces efficiency challenges due to, for example, the limited availability of cofactors like NAD+, which are essential for the catalytic activity of enzymes involved in this conversion. In this study, the enzyme CALDH (previously characterized from Gluconobacter oxydans DSM50049 genome) was purified and characterized to determine its cofactor and substrate specificity. CALDH was characterized to use HMF, FFCA, and furfural as substrates. The conversion of FFCA to FDCA and the catalytic ability of CALDH were assessed at different enzyme and substrate concentrations. Additionally, a system for NAD+ regeneration with Nox (NADH oxidase) was assayed, which improved FDCA yield threefold. The results demonstrated that the cofactor regeneration system significantly improved the yield of FDCA, offering a potential solution for the production of bio-based plastics. (Less)
Popular Abstract
The environmental impact of fossil-based plastics has become increasingly alarming, with global plastic production climbing from 370 million tonnes in 2018 to 400 million tonnes in 2022. Despite the urgent need for change, fossil-based plastics still dominate the market, accounting for 90.6% of total production in 2022, while bio-based plastics represent only a tiny fraction at 0.5%. The small amount of bioplastics highlights the vast potential and necessity for expanding the development of sustainable alternatives. One particularly promising biobased material is polyethylene furanoate (PEF), which is produced from 2,5-furandicarboxylic acid (FDCA). Unlike traditional PET, PEF offers better barrier properties and is recyclable, making it... (More)
The environmental impact of fossil-based plastics has become increasingly alarming, with global plastic production climbing from 370 million tonnes in 2018 to 400 million tonnes in 2022. Despite the urgent need for change, fossil-based plastics still dominate the market, accounting for 90.6% of total production in 2022, while bio-based plastics represent only a tiny fraction at 0.5%. The small amount of bioplastics highlights the vast potential and necessity for expanding the development of sustainable alternatives. One particularly promising biobased material is polyethylene furanoate (PEF), which is produced from 2,5-furandicarboxylic acid (FDCA). Unlike traditional PET, PEF offers better barrier properties and is recyclable, making it an ideal candidate for the next generation of sustainable plastics.
The parent molecule for FDCA is 5-hydroxymethylfurfural (HMF), which is obtained by dehydration of fructose or glucose, the components of sugar. Transformation of HMF to FDCA requires a series of 3 oxidation reactions that can be achieved by chemical or enzymatic catalysis. Chemical catalysis using noble metal catalysts is not environment friendly. On the other hand, enzymatic catalysis occurs under mild conditions but needs help of cofactors for transfer of electrons. The cofactor needs to be regenerated for its repeated use. A commonly used cofactor in living systems is Nicotinamide adenine dinucleotide (Phosphate) oxidized form (NAD(P)+) that is reduced to Nicotinamide adenine dinucleotide (Phosphate) reduced form (NAD(P)H) during electron transfer.
Earlier research at Division of Biotechnology, Lund University has shown that bacteria called Gluconobacter oxydans catalyses two of the three oxidation steps from HMF to FDCA. This thesis focuses on characterizing the enzyme Coniferyl Aldehyde Dehydrogenase (CALDH) from Gluconobacter oxydans that catalyses the last oxidation step for transforming 5-formyl-2-furancarboxylic acid (FFCA) to FDCA and studying the effect of cofactor regeneration using another enzyme NADH oxidase. The enzyme was purified and its activity with different substrates and cofactors (NAD+/NADP+) studied. Additionally, a new system was developed to recycle NAD+ and improved FDCA yield threefold. The results are promising, showing that this cofactor regeneration system could greatly enhance the efficiency of producing FDCA on a larger scale. (Less)
Please use this url to cite or link to this publication:
author
Li, Chengsi LU
supervisor
organization
course
KBTM01 20241
year
type
H2 - Master's Degree (Two Years)
subject
keywords
5-hydroxymethylfurfural, 2, 5-furandicarboxylic acid, NADH oxidase, Coniferyl aldehyde dehydrogenase, biotransformation, cofactor regeneration, bio-based plastics, biotechnology
language
English
id
9172811
date added to LUP
2024-08-30 15:40:24
date last changed
2024-08-30 15:40:24
@misc{9172811,
  abstract     = {{This thesis focuses on the characterization of Coniferyl aldehyde dehydrogenase (CALDH) and cofactor regeneration involved in the biotransformation process from 5-Formyl-2-furancarboxylic Acid (FFCA) to 2,5-furandicarboxylic acid (FDCA) using NADH oxidases. FDCA is a building block of bio-based polymers like polyethylene furanoate (PEF), which serves as a sustainable alternative to traditional Polyethylene terephthalate (PET) plastics. However, the biocatalytic process faces efficiency challenges due to, for example, the limited availability of cofactors like NAD+, which are essential for the catalytic activity of enzymes involved in this conversion. In this study, the enzyme CALDH (previously characterized from Gluconobacter oxydans DSM50049 genome) was purified and characterized to determine its cofactor and substrate specificity. CALDH was characterized to use HMF, FFCA, and furfural as substrates. The conversion of FFCA to FDCA and the catalytic ability of CALDH were assessed at different enzyme and substrate concentrations. Additionally, a system for NAD+ regeneration with Nox (NADH oxidase) was assayed, which improved FDCA yield threefold. The results demonstrated that the cofactor regeneration system significantly improved the yield of FDCA, offering a potential solution for the production of bio-based plastics.}},
  author       = {{Li, Chengsi}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Characterization of Coniferyl Aldehyde Dehydrogenase and cofactor regeneration with NADH oxidase in the biotransformation from FFCA to FDCA}},
  year         = {{2024}},
}