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Expression and characterization of a putative mannosylglucose phosphorylase from a human gut bacterium

Wiemann, Mathias LU (2017) KEMT10 20171
Department of Chemistry
Abstract
The human gut contains a vast number of bacteria. These bacteria allow us to digest the complex glycans that are part of our diet but we ourselves cannot digest. Among these bacteria one of the genera that is a major constituent is Bacteroidetes. One of the distinguishing features of this genus is its polysaccharide utilizing loci (PULs), clusters of genes for degradation of specific glycans and each cluster regulated by a common regulator. What PULs that are available differs between species, which leads to different bacteria being able to utilize different glycans. The bacteria Bacteroides ovatus has several PULs dedicated to the degradation of glycans containing mannan, a major constituent of hemicellulose in softwoods but also an... (More)
The human gut contains a vast number of bacteria. These bacteria allow us to digest the complex glycans that are part of our diet but we ourselves cannot digest. Among these bacteria one of the genera that is a major constituent is Bacteroidetes. One of the distinguishing features of this genus is its polysaccharide utilizing loci (PULs), clusters of genes for degradation of specific glycans and each cluster regulated by a common regulator. What PULs that are available differs between species, which leads to different bacteria being able to utilize different glycans. The bacteria Bacteroides ovatus has several PULs dedicated to the degradation of glycans containing mannan, a major constituent of hemicellulose in softwoods but also an important structural and energy-storage element in the food we digest.
One of these PULs from B. ovatus, containing ten genes, is up-regulated when it is grown on LBG galactomannan. Our group has earlier characterized three of the proteins encoded by these genes, showing that they are able to degrade different types of mannan containing glycans. We have also shown that this PUL is essential for B. ovatus to be able to grow galactomannan.
This work has been focused on the enzyme thought to be the final stage of the degradation, a mannsoylglucose phosphorylase (MGP) part of the glycoside hydrolase 130 (GH130) family. The gene encoding the enzyme has been cloned and expressed using a two-step cloning strategy, purified through both batch and column purification. The enzyme has been confirmed to be the correct protein through peptide mass fingerprinting (PMF) and we have shown that it is likely is a trimer through dynamic light scattering (DLS). The activity of the enzyme has been confirmed through the release of phosphate in the synthetic reaction, measured through a malachite green assay, and the creation of a disaccharide has been shown with mass spectrometry (MS). The build-up of the disaccharide over time was confirmed by time-point analysis of the reaction mixture using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). (Less)
Popular Abstract (Swedish)
Människan lever i samspel med en kolossal mängd bakterier. De finns på vår hud, i våra munnar och i våra tarmar. Detta samspel är en viktig del i vårat liv eftersom de bakterier som finns i våra tarmar har en viktig roll i nedbrytning av den näring vi får i oss. Dessa bakterier kan bryta ner olika typer av långa sockerkedjor som finns i växter som vi själva inte kan och låter oss därmed använda mer av den föda vi intar.Olika bakterier kan utnyttja olika typer av sockerkedjor. En av de bakterier som finns i våra tarmar är Bacteroides ovatus, en bakterie som kan bryta ner långa kedjor uppbygda av en sockertyp som kallas mannan. För att kunna göra det kan den tillverka en mängd olika proteiner som bryter ner mannan sockerkedjorna till mindre... (More)
Människan lever i samspel med en kolossal mängd bakterier. De finns på vår hud, i våra munnar och i våra tarmar. Detta samspel är en viktig del i vårat liv eftersom de bakterier som finns i våra tarmar har en viktig roll i nedbrytning av den näring vi får i oss. Dessa bakterier kan bryta ner olika typer av långa sockerkedjor som finns i växter som vi själva inte kan och låter oss därmed använda mer av den föda vi intar.Olika bakterier kan utnyttja olika typer av sockerkedjor. En av de bakterier som finns i våra tarmar är Bacteroides ovatus, en bakterie som kan bryta ner långa kedjor uppbygda av en sockertyp som kallas mannan. För att kunna göra det kan den tillverka en mängd olika proteiner som bryter ner mannan sockerkedjorna till mindre bitar som kan användas.

Detta arbetes syfte har varit att ta fram och undersöka ett av dessa proteiner. Proteinet, kallat BoMGP, är ett enzym som bryter ner sockermolekyler som består av ett mannos-socker och ett glukos-socker genom att fästa en fosfatmolekyl till mannos-sockret, ett så kallat fosforylas. Det som gör fosforylaserna speciella i jämförelse med andra socker-nedbrytande enzymer är att den även kan utföra den motsatta reaktionen. Det vill säga det kan det slå samman ett mannos-socker med en fosfat fäst till sockret och ett glukos-socker för att bygga en sockermolekyl som består av ett mannos-socker och ett glukos-socker samtidigt som det fosfatet frigörs från mannos-sockret. De flesta andra typer av socker-nedbrytande enzymer kan bara bryta ner och inte bygga upp.
Under projektet har vi gentekniskt producerat BoMGP, analyserat och bekräftat att det är rätt protein vi fått fram samt undersökt om det har den funktion som vi trodde att det hade när projektet började. Vi kan konstatera att det är rätt protein och att det verkar som om proteinet har den funktion som vi trodde.
Proteinet är intressant då det skulle kunna vara ytterligare ett verktyg för tillverkning av olika typer av molekyler inom så kallad grön kemi, kemi där man försöker ha så låg miljöpåverkan som möjligt. (Less)
Please use this url to cite or link to this publication:
author
Wiemann, Mathias LU
supervisor
organization
course
KEMT10 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Glycoside phosphorylase, GH130, Human gut bacteria, protein science, proteinvetenskap
language
English
id
8922567
date added to LUP
2017-08-18 13:20:48
date last changed
2017-08-18 13:20:48
@misc{8922567,
  abstract     = {The human gut contains a vast number of bacteria. These bacteria allow us to digest the complex glycans that are part of our diet but we ourselves cannot digest. Among these bacteria one of the genera that is a major constituent is Bacteroidetes. One of the distinguishing features of this genus is its polysaccharide utilizing loci (PULs), clusters of genes for degradation of specific glycans and each cluster regulated by a common regulator. What PULs that are available differs between species, which leads to different bacteria being able to utilize different glycans. The bacteria Bacteroides ovatus has several PULs dedicated to the degradation of glycans containing mannan, a major constituent of hemicellulose in softwoods but also an important structural and energy-storage element in the food we digest.
One of these PULs from B. ovatus, containing ten genes, is up-regulated when it is grown on LBG galactomannan. Our group has earlier characterized three of the proteins encoded by these genes, showing that they are able to degrade different types of mannan containing glycans. We have also shown that this PUL is essential for B. ovatus to be able to grow galactomannan.
This work has been focused on the enzyme thought to be the final stage of the degradation, a mannsoylglucose phosphorylase (MGP) part of the glycoside hydrolase 130 (GH130) family. The gene encoding the enzyme has been cloned and expressed using a two-step cloning strategy, purified through both batch and column purification. The enzyme has been confirmed to be the correct protein through peptide mass fingerprinting (PMF) and we have shown that it is likely is a trimer through dynamic light scattering (DLS). The activity of the enzyme has been confirmed through the release of phosphate in the synthetic reaction, measured through a malachite green assay, and the creation of a disaccharide has been shown with mass spectrometry (MS). The build-up of the disaccharide over time was confirmed by time-point analysis of the reaction mixture using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).},
  author       = {Wiemann, Mathias},
  keyword      = {Glycoside phosphorylase,GH130,Human gut bacteria,protein science,proteinvetenskap},
  language     = {eng},
  note         = {Student Paper},
  title        = {Expression and characterization of a putative mannosylglucose phosphorylase from a human gut bacterium},
  year         = {2017},
}