Random Mutagenesis on a De-novo enzyme : increasing the hydrolase activity of a Polycarbonate degrading enzyme.
Author
Term
4. semester
Education
Publication year
2023
Submitted on
2023-11-21
Pages
63
Abstract
The aim is to experimentally increase the hydrolase activity of a given polycarbonate degrading enzyme by directed evolution. 206 candidates were picked for screening from the provided library. During the course of this project 4 screening were done and the expressed proteins are screened for their activity on long chain and short chain fatty acid esters, using tributyrin and olive oil plate assays at 25°C and 37°C for 10 days. The variants were evaluated based on three criteria: absence of background activity and the order of appearance of hydrolase activity compared to the controls. Restriction enzyme analysis was performed to ensure the pres- ence of translation start codon in the plasmid DNA of variants showing ac- tivity. Out of 206, there are 5 interest- ing candidates 6A,11B,11C, 20I, and 18E, having hydrolase activity at 25°C.
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