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Cloning, expression and characterisation of Amidase Genes from a psychrotolerant Nesterenkonia isolate

<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.&nbsp / This study aimed to identify and characterize amidase genes from this novel psychrotolerant microorganism. Using BLAST analysis, two ORFs with conserved amidase sequences were&nbsp / 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&nbsp / family, respectively. On the genome, the spatial orientation and intergenic distance (1bp overlap) of the ORF‟s 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 &plusmn / 500bps downstream from amiS, suggesting the possible presence of a multi amidase&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / 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>
Date January 2009
CreatorsKwon, Hanna
Source SetsSouth African National ETD Portal
Detected LanguageEnglish
TypeThesis and dissertation
RightsCopyright: University of the Western Cape

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