Random Mutagenesis on a De-novo enzyme : increasing the hydrolase activity of a Polycarbonate degrading enzyme.
Author
Abdullah, Hira
Term
4. semester
Education
Publication year
2023
Submitted on
2023-11-21
Pages
63
Abstract
Formålet var at øge hydrolaseaktiviteten i et polycarbonat-nedbrydende enzym ved hjælp af rettet evolution (laboratorierunder med mutation og selektion). Fra et bibliotek af varianter blev 206 kandidater udvalgt og testet i fire screeningsrunder. De udtrykte proteiner blev afprøvet på kort- og langkædede fedtsyreestere ved hjælp af pladetests med tributyrin og olivenolie, som viser klare zoner, når estere nedbrydes. Inkubation foregik ved 25°C og 37°C i op til 10 dage. Varianterne blev vurderet på fravær af baggrundsaktivitet og hvor hurtigt hydrolaseaktivitet (spaltning af esterbindinger ved at tilsætte vand) dukkede op sammenlignet med kontroller. For varianter med aktivitet blev plasmid-DNA analyseret med restriktionsenzymer for at bekræfte, at startkodonet for translation var til stede. Ud af 206 viste fem varianter—6A, 11B, 11C, 20I og 18E—hydrolaseaktivitet ved 25°C.
This project aimed to increase the hydrolase activity of a polycarbonate-degrading enzyme using directed evolution (lab-based rounds of mutation and selection). From a library of variants, 206 candidates were chosen and tested across four screening rounds. The expressed proteins were assayed on short- and long-chain fatty acid esters using tributyrin and olive oil plate assays, which reveal clear zones when esters are broken down. Incubations were carried out at 25°C and 37°C for up to 10 days. Variants were evaluated for lack of background activity and for how quickly hydrolase activity (breaking ester bonds by adding water) appeared compared with controls. For active variants, plasmid DNA was examined with restriction enzyme analysis to confirm the presence of the translation start codon. Of the 206 tested, five variants—6A, 11B, 11C, 20I, and 18E—showed hydrolase activity at 25°C.
[This summary has been rewritten with the help of AI based on the project's original abstract]
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