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The kinetic characterization of mutated cyanobacterial dual-functioning fructose/sedoheptulose bisphosphate using a malachite green colorimetric assay

Jousif Anis, Albert LU (2021) KMBM05 20211
Applied Microbiology
Abstract
The increased human dependence on fossil-based fuels, industrial chemicals, and structural materials has caused a drastic increase in atmospheric CO2 levels during the past 50 years. Consequences of this have been observed in the disruption of terrestrial and aquatic ecosystems. Bioengineering of photosynthetic organisms capable of converting CO2 into biomass and value-added organic molecules provide a promising solution to the problem. However, faster CO2 fixation rates are needed in order to meet industrial scale production capacities. Previously published studies have mainly focused on the overexpression of Calvin cycle enzymes or engineering of ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) whereas fewer have looked into the... (More)
The increased human dependence on fossil-based fuels, industrial chemicals, and structural materials has caused a drastic increase in atmospheric CO2 levels during the past 50 years. Consequences of this have been observed in the disruption of terrestrial and aquatic ecosystems. Bioengineering of photosynthetic organisms capable of converting CO2 into biomass and value-added organic molecules provide a promising solution to the problem. However, faster CO2 fixation rates are needed in order to meet industrial scale production capacities. Previously published studies have mainly focused on the overexpression of Calvin cycle enzymes or engineering of ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) whereas fewer have looked into the engineering of enzymes downstream of RuBisCO. In this thesis, a malachite green-based colorimetric assay was optimized and used in characterizing the kinetic properties of wild type bifunctional cyanobacterial fructose/sedoheptulose bisphosphatase (cy-F/SBPase) and variants of the enzyme with respect to the hydrolysis of one of its substrates (fructose 1,6-bisphosphate). The assay was validated by successfully assaying a set of previously kinetically characterized cy-F/SBPase variants as well as the wild type. Subsequently, a group of rationally de novo designed mutants of the enzyme was kinetically characterized. This way, a crude understanding of new aspects of the protein structure-function relationship was established, which could potentially guide future protein engineering efforts on cy-F/SBPase. The developed malachite green assay could also complement such research efforts as a result of its multiplexed format which is desirable when assaying larger protein libraries. (Less)
Popular Abstract (Swedish)
De årliga koldioxidutsläppen har under de senaste 50 åren ökat med nästan 200% som påföljd av människans användning av fossila bränslen och råvaror. De negativa konsekvenserna av denna ökning syns idag världen över i land- och havsbaserade ekosystem, samt inom den biologiska mångfalden. Forskningens rön visar lovande lösningar genom bioteknisk ingenjörskonst av fotosyntetiska organismer med förmågan att ta upp och konvertera koldioxid till biomassa och förädlade kemikalier. Trots att dessa resultat ser lovande ut behövs mer forskning för att skala upp denna process i en industriell miljö. I detta examensarbete undersöks ett metaboliskt enzym som är involverat i att påskynda fotosyntesen. Målet var huvudsakligen att optimera och testa en... (More)
De årliga koldioxidutsläppen har under de senaste 50 åren ökat med nästan 200% som påföljd av människans användning av fossila bränslen och råvaror. De negativa konsekvenserna av denna ökning syns idag världen över i land- och havsbaserade ekosystem, samt inom den biologiska mångfalden. Forskningens rön visar lovande lösningar genom bioteknisk ingenjörskonst av fotosyntetiska organismer med förmågan att ta upp och konvertera koldioxid till biomassa och förädlade kemikalier. Trots att dessa resultat ser lovande ut behövs mer forskning för att skala upp denna process i en industriell miljö. I detta examensarbete undersöks ett metaboliskt enzym som är involverat i att påskynda fotosyntesen. Målet var huvudsakligen att optimera och testa en färgbaserad detektionsmetod som kan mäta den enzymatiska aktiviteten. Därefter mättes den enzymatiska aktiviteten av en rad nyskapade varianter av enzymet för att undersöka hur individuella fragment inom enzymet påverkar den katalytiska förmågan. Studiens resultat lyckades erhålla en grundläggande förståelse för hur vissa fragment påverkar enzymets katalysförmåga, som i sin tur skulle kunna tillämpas i framtida experiment inom enzymoptimering. (Less)
Please use this url to cite or link to this publication:
author
Jousif Anis, Albert LU
supervisor
organization
course
KMBM05 20211
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Cyanobacteria, carbon fixation, metabolic engineering, protein engineering, colorimetric enzyme assay, rational design, enzyme kinetics, applied microbiology
language
English
id
9056594
date added to LUP
2021-06-28 11:53:16
date last changed
2022-01-01 03:40:04
@misc{9056594,
  abstract     = {{The increased human dependence on fossil-based fuels, industrial chemicals, and structural materials has caused a drastic increase in atmospheric CO2 levels during the past 50 years. Consequences of this have been observed in the disruption of terrestrial and aquatic ecosystems. Bioengineering of photosynthetic organisms capable of converting CO2 into biomass and value-added organic molecules provide a promising solution to the problem. However, faster CO2 fixation rates are needed in order to meet industrial scale production capacities. Previously published studies have mainly focused on the overexpression of Calvin cycle enzymes or engineering of ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) whereas fewer have looked into the engineering of enzymes downstream of RuBisCO. In this thesis, a malachite green-based colorimetric assay was optimized and used in characterizing the kinetic properties of wild type bifunctional cyanobacterial fructose/sedoheptulose bisphosphatase (cy-F/SBPase) and variants of the enzyme with respect to the hydrolysis of one of its substrates (fructose 1,6-bisphosphate). The assay was validated by successfully assaying a set of previously kinetically characterized cy-F/SBPase variants as well as the wild type. Subsequently, a group of rationally de novo designed mutants of the enzyme was kinetically characterized. This way, a crude understanding of new aspects of the protein structure-function relationship was established, which could potentially guide future protein engineering efforts on cy-F/SBPase. The developed malachite green assay could also complement such research efforts as a result of its multiplexed format which is desirable when assaying larger protein libraries.}},
  author       = {{Jousif Anis, Albert}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{The kinetic characterization of mutated cyanobacterial dual-functioning fructose/sedoheptulose bisphosphate using a malachite green colorimetric assay}},
  year         = {{2021}},
}