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Integration of high-throughput specificity profiling and engineering of xylan-active enzymes and xylanosomes

(2023)
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(UGent) , (UGent) , (UGent) and (UGent)
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Abstract
The depletion of fossil fuels and the accumulation of greenhouse gases leading to global warming and climate change call for the implementation of sustainable alternative processes for obtaining energy and fossil fuel-dependent compounds. Lignocellulosic biomasses rich in stored energy and building blocks for high-value compounds can be obtained from agricultural and forestry residues, food processing industries and other industrial activities. Agricultural residues and by-products from cereals production like rice, wheat and barley contain considerable amounts of arabinoxylan (AX). AX degradation products, (arabino)xylan-oligosaccharides ((A)XOS), have been pointed out to be beneficial for human health due to their prebiotic properties, for example. The efficient enzymatic degradation of AX demands enzymes with characterized substrate specificities and known activity conditions. Metagenomic approaches are a widespread tool to dig into the immeasurable diversity of enzymes that are present in nature. However, the challenge is to have high-throughput (HT) approaches that can characterize the large set of enzyme candidates that result from these metagenomic studies. With this work, I aimed to contribute to the acceleration of the characterization and engineering of enzymes that can improve the degradation of the AX polymers. This has resulted in three main achievements. (1) The development of a HT assay for the specificity profiling of AX-acting enzymes using the DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). (2) For an optimal visualization of the large DSA-FACE data set, product profiles revealing enzymatic degradation maps were introduced. (3) Funnel-type pipelines consisting of parallel assays for the engineering and analysis of AX-acting enzyme complexes (Designer Xylanosomes (DXs)), inspired by the naturally occurring cellulosomes, were developed. The methodological approaches developed in this thesis have the objective of expediting the discovery of AX-acting enzymes and DXs possessing remarkable properties. Additionally, they serve as a scanning practice to fine-tune enzymes (complexes) for dedicated applications in the biorefinery industries.

Citation

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MLA
de Carvalho Maurício da Fonseca, Maria João. Integration of High-Throughput Specificity Profiling and Engineering of Xylan-Active Enzymes and Xylanosomes. Ghent University. Faculty of Bioscience Engineering, 2023.
APA
de Carvalho Maurício da Fonseca, M. J. (2023). Integration of high-throughput specificity profiling and engineering of xylan-active enzymes and xylanosomes. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Chicago author-date
Carvalho Maurício da Fonseca, Maria João de. 2023. “Integration of High-Throughput Specificity Profiling and Engineering of Xylan-Active Enzymes and Xylanosomes.” Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
Chicago author-date (all authors)
de Carvalho Maurício da Fonseca, Maria João. 2023. “Integration of High-Throughput Specificity Profiling and Engineering of Xylan-Active Enzymes and Xylanosomes.” Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
Vancouver
1.
de Carvalho Maurício da Fonseca MJ. Integration of high-throughput specificity profiling and engineering of xylan-active enzymes and xylanosomes. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2023.
IEEE
[1]
M. J. de Carvalho Maurício da Fonseca, “Integration of high-throughput specificity profiling and engineering of xylan-active enzymes and xylanosomes,” Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium, 2023.
@phdthesis{01H1CD9RZWB4SRAT2CAZHNXCFW,
  abstract     = {{The depletion of fossil fuels and the accumulation of greenhouse gases leading to global warming and climate change call for the implementation of sustainable alternative processes for obtaining energy and fossil fuel-dependent compounds. Lignocellulosic biomasses rich in stored energy and building blocks for high-value compounds can be obtained from agricultural and forestry residues, food processing industries and other industrial activities. Agricultural residues and by-products from cereals production like rice, wheat and barley contain considerable amounts of arabinoxylan (AX). AX degradation products, (arabino)xylan-oligosaccharides ((A)XOS), have been pointed out to be beneficial for human health due to their prebiotic properties, for example. The efficient enzymatic degradation of AX demands enzymes with characterized substrate specificities and known activity conditions. Metagenomic approaches are a widespread tool to dig into the immeasurable diversity of enzymes that are present in nature. However, the challenge is to have high-throughput (HT) approaches that can characterize the large set of enzyme candidates that result from these metagenomic studies. With this work, I aimed to contribute to the acceleration of the characterization and engineering of enzymes that can improve the degradation of the AX polymers. This has resulted in three main achievements. (1) The development of a HT assay for the specificity profiling of AX-acting enzymes using the DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). (2) For an optimal visualization of the large DSA-FACE data set, product profiles revealing enzymatic degradation maps were introduced. (3) Funnel-type pipelines consisting of parallel assays for the engineering and analysis of AX-acting enzyme complexes (Designer Xylanosomes (DXs)), inspired by the naturally occurring cellulosomes, were developed. The methodological approaches developed in this thesis have the objective of expediting the discovery of AX-acting enzymes and DXs possessing remarkable properties. Additionally, they serve as a scanning practice to fine-tune enzymes (complexes) for dedicated applications in the biorefinery industries.}},
  author       = {{de Carvalho Maurício da Fonseca, Maria João}},
  isbn         = {{9789463576253}},
  language     = {{eng}},
  pages        = {{XXVIII, 268}},
  publisher    = {{Ghent University. Faculty of Bioscience Engineering}},
  school       = {{Ghent University}},
  title        = {{Integration of high-throughput specificity profiling and engineering of xylan-active enzymes and xylanosomes}},
  year         = {{2023}},
}