Screening and Characterization of Novel Ulvan Degrading Lyases
(2023) KEMR30 20232Department of Chemistry
- Abstract
- Macroalgae are found worldwide dominating the aquatic ecosystems and representing a vast still sporadically explored resource with significant potential for mitigating climate change, constitute new food alternatives, provide sustainably fuel, and possibly be involved in new pharmaceutical discoveries. Despite limited research attention, ulvan, an irregular sulphated polysaccharide found in green algae holds an immense potential for biotechnological applications. The aim of this thesis study was therefore to screen and investigate prioritized novel ulvan lyases from both metagenome targets and gene mining of Wenyingzhuangia fucanilytica genome. A novel ulvan lyase attributed to PL40 family, termed WF-PL40-V demonstrating activity on ulvan... (More)
- Macroalgae are found worldwide dominating the aquatic ecosystems and representing a vast still sporadically explored resource with significant potential for mitigating climate change, constitute new food alternatives, provide sustainably fuel, and possibly be involved in new pharmaceutical discoveries. Despite limited research attention, ulvan, an irregular sulphated polysaccharide found in green algae holds an immense potential for biotechnological applications. The aim of this thesis study was therefore to screen and investigate prioritized novel ulvan lyases from both metagenome targets and gene mining of Wenyingzhuangia fucanilytica genome. A novel ulvan lyase attributed to PL40 family, termed WF-PL40-V demonstrating activity on ulvan from different Ulva species and was selected as a major target enzyme for subsequent investigation. WF-PL40-V was active in the crude cell lysate of expression host and remained soluble and active after affinity purification at high concentration with no significant aggregation observed. The protein solubility as well as lack of possible oligomerization was confirmed by analytical size-exclusion chromatography (SEC). The generated AlphaFold model of WF-PL40-V analysis allowed identification of two protein domains, the N-terminal catalytic domain followed by C-terminal substrate binding domain. Investigated enzyme demonstrated activity optimum at 35°C and pH 8.5 and slight preference to Ulva sp., fine grade ulvan in presence of 10 mM CaCl2 and 25 mM NaCl. Enzyme kinetics of WF-PL40-V resulted in a Vmax of 0.15 ± 0.1 mM min-1, KM was 0.07 ± 0.06 mM and kcat was estimated to 47 s-1. The generated product pattern by WF-PL40-V was composed of different sizes of ulvan oligosaccharides analyzed via thin-layer chromatography and preparative SEC. Noteworthy, the product pattern of the investigated enzymes varied slightly when using different substrates. The reaction products of optimal substrate were subjected for NMR analysis. Bioinformatic analysis allowed prediction of WF-PL40-V to a CAZyme gene cluster 4 along with other genes encoding putative enzymes needed for ulvan full uptake by W. fucanilytica. Independent transcription of WF-PL40-V gene was also predicted. This thesis study provided screening of putative ulvan lyases generating characterization of a novel PL40 family lyase, however, more investigation needs to be conducted to understand the complicated degradation mechanism of ulvan polysaccharides and its promising biotechnological applications. (Less)
- Popular Abstract
- Emerging challenges regarding climate change are a most constant threat to today’s society. The increasing demand of combating climate change and finding new alternative sources of food, biofuel, and pharmaceuticals, has led to a growing interest for marine resources, to potentially address these needs. The aquatic environment forms the natural habitat of macroalgae often referred to as seaweed which are photosynthetic organisms that comprise the basis of the primary production in the aquatic world. Macroalgae plays a crucial role in the carbon cycle by harnessing the energy generated by the sun to transform carbon into organic material also known as biomass while releasing oxygen to the atmosphere. This nearly untouched aquatic biomass... (More)
- Emerging challenges regarding climate change are a most constant threat to today’s society. The increasing demand of combating climate change and finding new alternative sources of food, biofuel, and pharmaceuticals, has led to a growing interest for marine resources, to potentially address these needs. The aquatic environment forms the natural habitat of macroalgae often referred to as seaweed which are photosynthetic organisms that comprise the basis of the primary production in the aquatic world. Macroalgae plays a crucial role in the carbon cycle by harnessing the energy generated by the sun to transform carbon into organic material also known as biomass while releasing oxygen to the atmosphere. This nearly untouched aquatic biomass has huge potential for many biotechnological applications (Konasani et al., 2018).
There are three types of macroalgae, brown, red, and green which all contain a diverse range of carbohydrates. These carbohydrates are composted of linked sugar, many of which are rare, trapped in elongated chains called polysaccharides (Bäumgen, 2020). Thus far, research has been focused on red and brown algae, however, green algae constitute great potential as well. One of the predominant polysaccharides in green algae is called ulvan which is rare in other organisms. It is a water-soluble polysaccharide covered in sulphate groups (Tang et al., 2021). Marine bacteria, essential players in the carbon cycle, are involved in the degradation and regeneration of sequestered carbon in seaweed. These bacteria are capable of breaking down polysaccharides into shorter fragments, facilitating the uptake of energy to their cells. The aquatic bacteria have therefore evolved an intricate degradation system to break down polysaccharides using a set of polysaccharide degrading enzymes which enhance the speed of the degrading reactions (Garron et. al, 2020). The aim of this thesis was therefore to investigate several newly discovered ulvan degrading enzymes originated from marine bacteria.
In order to study the activity of these enzymes, the genes for the enzymes were cloned into Escherichia coli which is a common bacteria used in research to produce foreign proteins. The enzymes were cultivated in optimal condition and purified followed by activity testing. An ulvan degrading enzyme referred to as WF-PL40-V which is a member of a novel enzyme family, remained active after purification. This high-mass enzyme, WF-PL40-V, was prioritized and further characterized by determining the optimal reaction conditions for the enzyme to work efficiently. Different species of green algae was tested to investigate the optimal substrate for the enzyme. Furthermore, the tertiary-structure of the enzyme was investigated by analysing a computer model, indicated that the enzyme was most likely comprised of a single unit of two parts ensuring activity and recognition of substrate. Another aspect of the study was to understand what products that was generated by WF-PL40-V. This was evaluated by applying the product to various separation methods and full preparation of interesting product fraction for structural analysis. The thesis resulted in giving insight in the intricate degradation pathway of green algae by investigating a new enzyme family. However, more research needs to be conducted to understand the full degradation pathway of ulvan polysaccharides. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9142259
- author
- Malmgren, Annika LU
- supervisor
- organization
- course
- KEMR30 20232
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- macroalgae, ulvan, polysaccharide lyases, CAZymes, enzyme characterization, biochemistry
- language
- English
- id
- 9142259
- date added to LUP
- 2023-12-20 09:33:00
- date last changed
- 2023-12-20 09:33:00
@misc{9142259,
abstract = {{Macroalgae are found worldwide dominating the aquatic ecosystems and representing a vast still sporadically explored resource with significant potential for mitigating climate change, constitute new food alternatives, provide sustainably fuel, and possibly be involved in new pharmaceutical discoveries. Despite limited research attention, ulvan, an irregular sulphated polysaccharide found in green algae holds an immense potential for biotechnological applications. The aim of this thesis study was therefore to screen and investigate prioritized novel ulvan lyases from both metagenome targets and gene mining of Wenyingzhuangia fucanilytica genome. A novel ulvan lyase attributed to PL40 family, termed WF-PL40-V demonstrating activity on ulvan from different Ulva species and was selected as a major target enzyme for subsequent investigation. WF-PL40-V was active in the crude cell lysate of expression host and remained soluble and active after affinity purification at high concentration with no significant aggregation observed. The protein solubility as well as lack of possible oligomerization was confirmed by analytical size-exclusion chromatography (SEC). The generated AlphaFold model of WF-PL40-V analysis allowed identification of two protein domains, the N-terminal catalytic domain followed by C-terminal substrate binding domain. Investigated enzyme demonstrated activity optimum at 35°C and pH 8.5 and slight preference to Ulva sp., fine grade ulvan in presence of 10 mM CaCl2 and 25 mM NaCl. Enzyme kinetics of WF-PL40-V resulted in a Vmax of 0.15 ± 0.1 mM min-1, KM was 0.07 ± 0.06 mM and kcat was estimated to 47 s-1. The generated product pattern by WF-PL40-V was composed of different sizes of ulvan oligosaccharides analyzed via thin-layer chromatography and preparative SEC. Noteworthy, the product pattern of the investigated enzymes varied slightly when using different substrates. The reaction products of optimal substrate were subjected for NMR analysis. Bioinformatic analysis allowed prediction of WF-PL40-V to a CAZyme gene cluster 4 along with other genes encoding putative enzymes needed for ulvan full uptake by W. fucanilytica. Independent transcription of WF-PL40-V gene was also predicted. This thesis study provided screening of putative ulvan lyases generating characterization of a novel PL40 family lyase, however, more investigation needs to be conducted to understand the complicated degradation mechanism of ulvan polysaccharides and its promising biotechnological applications.}},
author = {{Malmgren, Annika}},
language = {{eng}},
note = {{Student Paper}},
title = {{Screening and Characterization of Novel Ulvan Degrading Lyases}},
year = {{2023}},
}