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Identifier

uch.biology.msc//2001koutsioulis

Title

Κατευθυνόμενη εξέλιξη δύο ενζυμικών μορίων

Author

Κουτσιούλης, Δημήτρης

Thesis advisor

Μπουριώτης, Βασίλης

Abstract

In the present study we employed directed evolution in order to improve selected properties of two enzymes: chitin deacetylase Cda2p from the zymomyces Saccharomyces cerevisiae and alkaline phosphatase from the Antarctic strain TAB5. The properties-targets for Cda2p were product inhibition by acetic acid and the incapability of the enzyme to act upon insoluble chitin substrates, while for TAB5AP the low thermostability. We applied error prone PCR on both genes and subsequently screened the mutated libraries. Unfortunately, the existing screening assays for Cda2p, didn't match with the needs of our experiments, so we were unable to screen the mutated clones. On the other hand we screened approximately 5500 clones from the TAB5AP libraries and selected 12 clones, which demonstrated higher levels of thermostability as compared to the wild type enzyme. Finally, we cloned and overexpressed the CDA2 gene in Escherichia coli. At the same time we designed two schemes of partial purification of the recombinant protein.

Language

Greek

Issue date

2001-12-06

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses

Type of Work--Post-graduate theses

Notes

Διατμηματικό, συνεργαζόμενα Τμήματα Βιολογίας και Ιατρικής

Permanent Link

https://elocus.lib.uoc.gr//dlib/4/a/2/metadata-dlib-2001koutsioulis.tkl