<p>A nitrile and amide hydrolysing Nesterenkonia sp. was isolated from Antarctic soil and was characterised as a psychrotolerant, halotolerant and alkaliphilic extremophile. Amidases are widely distributed in both prokaryotic and eukaryotic organisms. These enzymes hydrolyze C-N bonds other than peptide bonds and are particularly interesting for their potential industrial application.  / This study aimed to identify and characterize amidase genes from this novel psychrotolerant microorganism. Using BLAST analysis, two ORFs with conserved amidase sequences were  / identified from the complete genome sequence of the organism. Two ORFs, AmiF and AmiS, were assigned to two different gene families, the aceta/formamidase family and amidase signature  / family, respectively. On the genome, the spatial orientation and intergenic distance (1bp overlap) of the ORFâs suggested that amiF and amiS could possibly be cotranscribed which was confirmed by reverse transcription PCR. A third ORF with a conserved amidase sequence was found ± / 500bps downstream from amiS, suggesting the possible presence of a multi amidase  / operon. The two genes were cloned and expressed as N-terminal 6x His-Tag fusion proteins. AmiS and Ami F were partially purified using Ni-chelation chromatography. Although both proteins  / were subjected to activity assay, their activities are yet to be established. Homology modeling of the AmiF and AmiS translated sequences showed that the proteins had the significant similarities  / to the members of their families. Although the sequence identities between the AmiF and AmiS and their templates were very low (24 % and 25% respectively), the evaluation of the models  / showed that the quality of the models were good. This study reports the genetic and functional characterisation of amidase genes from the cold adapted microorganisms.</p>
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uwc/oai:UWC_ETD:http%3A%2F%2Fetd.uwc.ac.za%2Findex.php%3Fmodule%3Detd%26action%3Dviewtitle%26id%3Dgen8Srv25Nme4_9975_1363786362 |
| Date | January 2009 |
| Creators | Kwon, Hanna |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis and dissertation |
| Format | |
| Coverage | ZA |
| Rights | Copyright: University of the Western Cape |
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