Characterization of ulvan polysaccharide degrading enzymes from Wenyingzhuangia fucanilytica
(2022) KBTM05 20221Biotechnology
Biotechnology (MSc)
- Abstract
- The aim of this thesis was to fully characterize the enzyme WFp-105, including bioinformatic analysis, structural modeling, full activity and stability characterization and product determination. As well as for the enzyme WFp-37; optimize protein production in regard to expression strains and cultivation temperature, analyzing the genomic context as well as performing an initial activity detection.
From the genome of Wenyingzhuangia fucanilytica two enzymes were cloned, one glucoside hydrolase termed WFp-105 and one polysaccharide lyase termed WFp-37 attributed to polysaccharide lyase family 37. Both were heterologously produced in Escherichia coli and purified using affinity chromatography. WFp-37 showed activity on raw ulvan in the... (More) - The aim of this thesis was to fully characterize the enzyme WFp-105, including bioinformatic analysis, structural modeling, full activity and stability characterization and product determination. As well as for the enzyme WFp-37; optimize protein production in regard to expression strains and cultivation temperature, analyzing the genomic context as well as performing an initial activity detection.
From the genome of Wenyingzhuangia fucanilytica two enzymes were cloned, one glucoside hydrolase termed WFp-105 and one polysaccharide lyase termed WFp-37 attributed to polysaccharide lyase family 37. Both were heterologously produced in Escherichia coli and purified using affinity chromatography. WFp-37 showed activity on raw ulvan in the crude cell lysate. However, it was prone to aggregation as pure enzyme. The glucoside hydrolase attributed to glycoside hydrolase family 105 by gene phylogenetic analysis and structural modeling. Recombinant WFp-105 was purified to high concentrations and remained soluble. Enzyme demonstrated activity on unsaturated oligosaccharides from ulvan in the temperature range 10-40°C and pH 5-7.5. WFp-105 activity optima corresponded to algae containing ulvan growth conditions. Hydrolysis product detection with thin layer chromatography as well as product profiling with high-performance anion exchange chromatography - pulsed amperometric detection confirmed generation of monosaccharides from unsaturated oligosaccharides, in particular xylose, as well as three other products that was not successfully attributed to any mono- or oligosaccharide. Further investigation is needed in order to fully investigate potential of studied enzymes to be implemented for biotechnological applications. (Less) - Popular Abstract
- Climate change and eutrophication of seas and oceans, due to the overfertilization of farmlands, has given rise to an increase in seasonal algal blooms know as green tides. One proposed way to combat this problem is to cultivate fast-growing macro algae, or seaweed as it is also called, in affected areas and/or close to estuaries emerging from agricultural lands. The cultivated algae can then be used in several different applications from human food and animal feed to industrial applications such as cosmetics. The algae also contain rare sugar molecules that are not commonly found in plants grown on land, sugar molecules which are believed to possess several health benefits such as reducing inflammation and promoting good intestinal... (More)
- Climate change and eutrophication of seas and oceans, due to the overfertilization of farmlands, has given rise to an increase in seasonal algal blooms know as green tides. One proposed way to combat this problem is to cultivate fast-growing macro algae, or seaweed as it is also called, in affected areas and/or close to estuaries emerging from agricultural lands. The cultivated algae can then be used in several different applications from human food and animal feed to industrial applications such as cosmetics. The algae also contain rare sugar molecules that are not commonly found in plants grown on land, sugar molecules which are believed to possess several health benefits such as reducing inflammation and promoting good intestinal bacteria and gut health. However, they are locked in long chains of repeating sugar molecules, called polysaccharides, and need to be degraded. The best way to perform this degradation is with the use of enzymes. Enzymes are proteins that help chemical reactions, such as the ones that break down polysaccharides, and they are found in every living organism. Every enzyme catalyzes a specific reaction so to get a certain result several enzymes need to be investigated.
Two of these enzymes that degrade polysaccharides from algae was investigated in this report. They were isolated from the chromosome of the bacteria Wenyingzhuangia fucanilytica which was found in fermenting algae in shallow coastal waters. To be able to analyze these enzymes they first needed to be produced. This is done by modifying the genes of another bacteria, Escherichia coli, which is a bacteria that is easy to cultivate and fast growing in laboratory conditions. The gene modified cells are then grown and harvested. From the cells, the enzymes are then purified and characterized confirming activity.
One of the studied enzymes, which catalyze the first reaction in breaking down the polysaccharides, was active on ulvan, which is a polysaccharide derived from green seaweed, and could be introduced to raw polysaccharide treatment obtaining products of high additional value. The other enzyme, a glucoside hydrolase which catalyze the last reaction in breaking down the polysaccharides, was producible in a soluble form and catalyzes the reaction into the rare sugar molecules that are also of biotechnological interest. Both characterized enzymes were active at temperatures close to room temperature and were most active at mildly acidic conditions. Further investigation will allow to test both enzymes in polysaccharide ulvan disruption processes. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9097086
- author
- Apelqvist, Erik LU
- supervisor
- organization
- course
- KBTM05 20221
- year
- 2022
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Enzyme, Enzymatic characterization, Recombinant protein producaiton, Protien purification, E. coli, W. fucanilytica, Transformation, Overexpression, Seaweed, Ulvan, Polysaccharides, biotechnology
- language
- English
- id
- 9097086
- date added to LUP
- 2022-08-18 13:43:35
- date last changed
- 2022-08-18 13:43:35
@misc{9097086,
abstract = {{The aim of this thesis was to fully characterize the enzyme WFp-105, including bioinformatic analysis, structural modeling, full activity and stability characterization and product determination. As well as for the enzyme WFp-37; optimize protein production in regard to expression strains and cultivation temperature, analyzing the genomic context as well as performing an initial activity detection.
From the genome of Wenyingzhuangia fucanilytica two enzymes were cloned, one glucoside hydrolase termed WFp-105 and one polysaccharide lyase termed WFp-37 attributed to polysaccharide lyase family 37. Both were heterologously produced in Escherichia coli and purified using affinity chromatography. WFp-37 showed activity on raw ulvan in the crude cell lysate. However, it was prone to aggregation as pure enzyme. The glucoside hydrolase attributed to glycoside hydrolase family 105 by gene phylogenetic analysis and structural modeling. Recombinant WFp-105 was purified to high concentrations and remained soluble. Enzyme demonstrated activity on unsaturated oligosaccharides from ulvan in the temperature range 10-40°C and pH 5-7.5. WFp-105 activity optima corresponded to algae containing ulvan growth conditions. Hydrolysis product detection with thin layer chromatography as well as product profiling with high-performance anion exchange chromatography - pulsed amperometric detection confirmed generation of monosaccharides from unsaturated oligosaccharides, in particular xylose, as well as three other products that was not successfully attributed to any mono- or oligosaccharide. Further investigation is needed in order to fully investigate potential of studied enzymes to be implemented for biotechnological applications.}},
author = {{Apelqvist, Erik}},
language = {{eng}},
note = {{Student Paper}},
title = {{Characterization of ulvan polysaccharide degrading enzymes from Wenyingzhuangia fucanilytica}},
year = {{2022}},
}