Overexpression and secretion of PET-hydrolysing enzymes in Pseudomonas putida KT2440
Author
Porotti, Enrico
Term
4. term
Education
Publication year
2023
Submitted on
2023-12-20
Abstract
Enzymatic plastic hydrolysis is a promising alternative to energy-intensive chemical recycling. This thesis investigates whether Pseudomonas putida KT2440 can overexpress and secrete an improved PET-hydrolyzing enzyme variant (LCCICCG-KRP). A plasmid carrying LCCICCG-KRP fused to an N-terminal signal peptide for periplasmic localization was constructed and used to transform P. putida. No strong evidence of enzyme secretion was found, but LCCICCG-KRP was successfully purified from P. putida. PET depolymerization assays on amorphous PET showed that the enzyme produced in P. putida has activity comparable to LCCICCG-KRP expressed in E. coli, achieving around 20% weight loss over 72 hours at 70 °C while using less than 2% of the enzyme amount required in standardized assays. The work details vector assembly, transformations, expression studies, purification (including SDS-PAGE and protein quantification), and enzymatic activity tests, and discusses approaches to improve expression and engineer secretion of heterologous proteins in P. putida KT2440.
Plastikgenanvendelse ved hjælp af enzymer er et lovende alternativ til energitunge kemiske processer. Denne afhandling undersøger, om Pseudomonas putida KT2440 kan overudtrykke og udskille en forbedret PET-nedbrydende enzymvariant (LCCICCG-KRP). Et plasmid med LCCICCG-KRP og et N-terminalt signalpeptid til periplasmisk lokalisation blev konstrueret og brugt til at transformere P. putida. Der blev ikke fundet stærke beviser for udskillelse af enzymet, men LCCICCG-KRP blev succesfuldt renset fra P. putida. Depolymeriseringsforsøg på amorf PET viste, at enzymet produceret i P. putida har en aktivitet på niveau med LCCICCG-KRP udtrykt i E. coli, med ca. 20% vægttab over 72 timer ved 70 °C, og med mindre end 2% af enzymmængden, der normalt anvendes i standardiserede assays. Arbejdet beskriver desuden samling af udtryksvektorer, transformationer, udtryksstudier, oprensning (herunder SDS-PAGE og proteinmåling) og enzymatiske aktivitetstests, samt drøfter tilgange til at forbedre udtryk og engineering af udskillelse af heterologe proteiner i P. putida KT2440.
[This apstract has been generated with the help of AI directly from the project full text]
Keywords