Enzymatic conversion of galactosylated glycans by gut bacteria : Structural and functional characterisation of carbohydrate-active enzymes
(2025)- Abstract
- The human gut microbiome is a highly complex environment, where a myriad of intricate microbiota-microbiota and microbiota-host interactions occur. The human gut microbiota has been associated with both health and disease. The research presented in this thesis focuses on investigating how the butyrate-producing human gut bacterium Roseburia hominis A2-183 (R. hominis) processes β-mannan oligosaccharides, which are derived from plant cell wall β-mannan glycans and may hold prebiotic potential.
In Paper I, the ability of R. hominis to ferment linear and galactosylated β-mannan oligosaccharides (MOS/GMOS) was investigated. Co-cultures of R. hominis and the acetate producing gut bacterium Bifidobacterium adolescentis demonstrated... (More) - The human gut microbiome is a highly complex environment, where a myriad of intricate microbiota-microbiota and microbiota-host interactions occur. The human gut microbiota has been associated with both health and disease. The research presented in this thesis focuses on investigating how the butyrate-producing human gut bacterium Roseburia hominis A2-183 (R. hominis) processes β-mannan oligosaccharides, which are derived from plant cell wall β-mannan glycans and may hold prebiotic potential.
In Paper I, the ability of R. hominis to ferment linear and galactosylated β-mannan oligosaccharides (MOS/GMOS) was investigated. Co-cultures of R. hominis and the acetate producing gut bacterium Bifidobacterium adolescentis demonstrated beneficial cross-feeding of metabolites, leading to enhanced growth and butyrate production of R. hominis. Three key enzymes - an exo-mannosidase (RhMan113A), an β-mannoside phosphorylase (RhMOP130A), and a α-galactosidase (RhGal36A) - were recombinantly expressed and investigated for their potential roles in the initial processing of MOS/GMOS.
Paper II expanded the characterisation studies of the RhMosUL β-mannoside phosphorylase RhMOP130A and its reaction products. During incubations with MOS and α-D-mannose 1-phosphate (M1P), RhMOP130A was capable of generating elongated reaction products with a degree of polymerisation estimated up to 10 mannosyl-units or more. These oligosaccharides precipitated and formed non-crystalline particles during prolonged reactions under standard assay conditions. The glycosyl acceptor molecule can be either mannose- or glucose-based, allowing for the synthesis of customised and novel glycans. The crystal structure of RhMOP130A revealed a +2 subsite, which may facilitate the stabilisation of longer ligands and reaction products.
Paper III examined the differences between the RhMosUL-originating α-galactosidase RhGal36A and the distantly located RhGal36B. While both enzymes had low activity towards polymeric galactomannan, RhGal36A was approximately 460-times more efficient than RhGal36B in hydrolysing galactosylated β-mannan oligosaccharides (GMOS), suggesting GMOS as a natural substrate for RhGal36A. To investigate the role of the N-domain loop, a catalytically active mutant of RhGal36A was constructed with a partial loop deletion, resulting in a threefold and fivefold reduction in specific activity towards GMOS and raffinose, respectively. The crystal structure of RhGal36A revealed two putative binding subsites that may aid in stabilising longer GMOS, an ability which was noted during substrates incubations.
(Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/e436ffea-1d45-4dd3-909c-bb5c6efb737f
- author
- Majtorp, Lovisa LU
- supervisor
-
- Henrik Stålbrand LU
- Derek Logan LU
- opponent
-
- Professor Abou Hachem, Maher, DTU Technical University of Denmark
- organization
- publishing date
- 2025
- type
- Thesis
- publication status
- published
- subject
- keywords
- Glycoside hydrolases, Phosphorylases, α-galactosidases, Human gut bacteria, Enzyme engineering, Structural biology, HPEAC-PAD, MALDI-ToF, X-ray crystallography
- pages
- 282 pages
- defense location
- Kemicentrum Sal A
- defense date
- 2025-05-09 13:00:00
- ISBN
- 978-91-8096-100-4
- 978-91-8096-101-1
- project
- Enzymatic conversion of galactosylated glycans by gut bacteria
- language
- English
- LU publication?
- yes
- id
- e436ffea-1d45-4dd3-909c-bb5c6efb737f
- date added to LUP
- 2025-04-02 15:23:01
- date last changed
- 2025-04-11 09:29:07
@phdthesis{e436ffea-1d45-4dd3-909c-bb5c6efb737f,
abstract = {{The human gut microbiome is a highly complex environment, where a myriad of intricate microbiota-microbiota and microbiota-host interactions occur. The human gut microbiota has been associated with both health and disease. The research presented in this thesis focuses on investigating how the butyrate-producing human gut bacterium Roseburia hominis A2-183 (R. hominis) processes β-mannan oligosaccharides, which are derived from plant cell wall β-mannan glycans and may hold prebiotic potential.<br/>In Paper I, the ability of R. hominis to ferment linear and galactosylated β-mannan oligosaccharides (MOS/GMOS) was investigated. Co-cultures of R. hominis and the acetate producing gut bacterium Bifidobacterium adolescentis demonstrated beneficial cross-feeding of metabolites, leading to enhanced growth and butyrate production of R. hominis. Three key enzymes - an exo-mannosidase (RhMan113A), an β-mannoside phosphorylase (RhMOP130A), and a α-galactosidase (RhGal36A) - were recombinantly expressed and investigated for their potential roles in the initial processing of MOS/GMOS. <br/>Paper II expanded the characterisation studies of the RhMosUL β-mannoside phosphorylase RhMOP130A and its reaction products. During incubations with MOS and α-D-mannose 1-phosphate (M1P), RhMOP130A was capable of generating elongated reaction products with a degree of polymerisation estimated up to 10 mannosyl-units or more. These oligosaccharides precipitated and formed non-crystalline particles during prolonged reactions under standard assay conditions. The glycosyl acceptor molecule can be either mannose- or glucose-based, allowing for the synthesis of customised and novel glycans. The crystal structure of RhMOP130A revealed a +2 subsite, which may facilitate the stabilisation of longer ligands and reaction products. <br/>Paper III examined the differences between the RhMosUL-originating α-galactosidase RhGal36A and the distantly located RhGal36B. While both enzymes had low activity towards polymeric galactomannan, RhGal36A was approximately 460-times more efficient than RhGal36B in hydrolysing galactosylated β-mannan oligosaccharides (GMOS), suggesting GMOS as a natural substrate for RhGal36A. To investigate the role of the N-domain loop, a catalytically active mutant of RhGal36A was constructed with a partial loop deletion, resulting in a threefold and fivefold reduction in specific activity towards GMOS and raffinose, respectively. The crystal structure of RhGal36A revealed two putative binding subsites that may aid in stabilising longer GMOS, an ability which was noted during substrates incubations. <br/>}},
author = {{Majtorp, Lovisa}},
isbn = {{978-91-8096-100-4}},
keywords = {{Glycoside hydrolases; Phosphorylases; α-galactosidases; Human gut bacteria; Enzyme engineering; Structural biology; HPEAC-PAD; MALDI-ToF; X-ray crystallography}},
language = {{eng}},
school = {{Lund University}},
title = {{Enzymatic conversion of galactosylated glycans by gut bacteria : Structural and functional characterisation of carbohydrate-active enzymes}},
url = {{https://lup.lub.lu.se/search/files/212901346/Enzymatic_conversion_of_galactosylated_glycans_by_gut_bacteria_-_Lovisa_Majtorp.pdf}},
year = {{2025}},
}