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