Characterisation of a DNA ligase from an Antarctic metagenomic library

• Main Author: Booysen, Dean
• Other Authors: Cowan, D. A
• Format: Thesis
• Language: English
• Published: University of the Western Cape 2011
• Subjects: Metagenomic DNA; sequence based screening; NAD+-dependent DNA ligase; ligase; Antarctica; bioinformatics; Antarctic; Miers; Miers Valley; Antarc*
• Online Access: http://hdl.handle.net/11394/4003

>Magister Scientiae - MSc A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations