Post-graduate theses
Current Record: 22 of 800
|
|
| Identifier |
000460515 |
| Title |
Overexpression, purification and biochemical characterization of native Parhyale hawaiensis glycosyl hydrolases |
| Alternative Title |
Υπερέκφραση, καθαρισμός και βιοχημικός χαρακτηρισμός ενδογενών ενζύμων διάσπασης κυτταρίνης του οργανισμού Parhyale hawaiensis |
|
Author
|
Καραπιδάκη, Ειρήνη Αρετή Ν.
|
|
Thesis advisor
|
Παυλόπουλος, Αναστάσιος
|
|
Reviewer
|
Γκουρίδης, Γεώργιος
Παυλίδης, Ιωάννης
|
| Abstract |
Cellulose is the main component of lignocellulosic plant biomass presenting an abundant and
renewable resource for biofuel production and various biotechnological applications.
However, the compact, crystalline structure and chemical composition of lignocellulose make
it resistant to chemical breakdown. To address this limitation, the biofuel industry utilizes
enzymatic cocktails produced by living organisms to promote the degradation of cellulose into
fermentable sugars. Recent research in certain marine crustaceans feeding on wood has
revealed that they encode in their genome all the necessary enzymes for extracting sugars
from cellulose. Remarkably, unlike most other animals, these organisms do not depend on
symbiotic microorganisms. Among these marine crustaceans, the genetically tractable
Parhyale hawaiensis provides an excellent model to investigate autonomous cellulose
biodegradation in a simple digestive gland. My Master thesis research concentrated on the
analysis of Parhyale glycosyl hydrolase genes from the GH7 family, known as
cellobiohydrolases or exoglucanases, that are uniquely present in crustaceans among
metazoans. First, a gain-of-function strategy was devised to explore the activity of an in vivo
overexpressed, tagged GH7 protein in cellulose digestion. This approach involved the
generation of stable Parhyale transgenic lines using the Minos transposable element, in
combination with a heat-inducible system for conditional overexpression of Parhyale GH7.
Upon heat-shock, a significant increase in cellulolytic activity was observed with the
dinitrosalicylic acid assay, suggesting that overexpressed GH7 enhances the cellulolytic
capacity of the enzymatic cocktail in Parhyale digestive glands. Second, the overexpressed,
tagged GH7 enzyme was affinity purified and activity assays with the cellobiohydrolase-specific
p-nitrophenyl-β-D-cellobioside substrate confirmed its functionality. Comparisons between
the Parhyale GH7 and a commercial, recombinant, fungal cellobiohydrolase indicated that
they exhibit comparable activities. Finally, a CRISPR non homologous end joining knock-in
method was employed for midgut-specific expression of the tagged Parhyale GH7
cellobiohydrolase, further expanding the prospects for understanding and utilizing these
enzymes. This research represents the first integrated genetic and biochemical study of the
autonomous cellulolytic capacity of Parhyale that opens up various avenues for future
investigations and potential biotechnological applications.
|
| Language |
English |
| Subject |
Affinity purification |
|
Biochemical assays. |
|
CRISPR NHEJ knock-in |
|
Cellobiohydrolase/exoglucanase GH7 |
|
Cellulose digestion |
|
Heat-inducible gene overexpression |
|
Βιοχημική ανάλυση |
|
Εξωγλουκανάση GH7 |
|
Θερμοεπαγώμενη υπερέκφραση γονιδίων |
|
Πρωτεϊνικός καθαρισμός |
| Issue date |
2023-11-24 |
| Collection
|
School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
|
|
|
Type of Work--Post-graduate theses
|
| Permanent Link |
https://elocus.lib.uoc.gr//dlib/f/4/7/metadata-dlib-1700655066-869960-12590.tkl
|
| Views |
886 |
Collections
