Purification, characterization and in-silico analysis of nitrilase from Gordonia terrae.

An inducible and aromatic nitrilase from Gordonia terrae was purified with a yield of 19%. The enzyme had turnover number of 63 s⁻¹ x 10⁻¹, Km 1.4 mM and Vmax 95 Umg⁻¹ protein for benzonitrile. The nitrilase of G. terrae was active at basic pH (7-10), moderate temperature (20-45 °C) and has a half-life of 4 h at 35 °C. MALDI analysis and amino acid sequence deduced from cloned nucleotide fragment showed 97% homology with putative amidohydrolase of Gordonia sputi NBRC 100414 and G. namibiensis. The enzyme showed regioselectivity towards hydroxybenzonitriles, as different position of hydroxyl group i.e. meta-, para- and orthosubstitutions on benzonitrile effect enzyme activity. The in-silico interactions of these substrates with the predicted 3D model of this enzyme also showed differential interaction between hydroxyl group of substrates and the polar amino acids surrounding enzyme's active site. This leads to different proximity and orientation of substrates vis-a-vis their interaction with catalytic residues.

Molecular detection and phylogenetic analysis of the catechol 1,2-dioxygenase gene from Gordonia spp.

The C12O gene (catA gene) encodes for catechol 1,2-dioxygenase, which is a key enzyme involved in the first step catalysis of the aromatic ring in the ortho-cleavage pathway. This functional gene can be used as a marker to assess the catabolic potential of bacteria in bioremediation. C12OF and C12OR primers were designed based on the conserved regions of the CatA amino acid sequence of Actinobacteria for amplifying the catA gene from the genus Gordonia (16 Gordonia representing 11 species). The amplified catA genes (382bp) were sequenced and analyzed. In the phylogenetic tree based on the translated catA amino acid sequences, all the Gordonia segregated clearly from other closely related genera. The sequence similarity of the catA gene in Gordonia ranged from 72.4% to 99.5%, indicating that the catA gene might have evolved faster than rrn operons or the gyrB gene at the inter-species level. A single nucleotide deletion of the catA gene was observed in Gordonia amicalis CC-MJ-2a, Gordonia rhizosphera and Gordonia sputi at nucleotide position 349. This deletion led to an encoding frame shift downstream of 11 amino acid residues, from WPSVAARAPAP to GHPWRPAHLHL, which was similar to most of the non-Gordonia Actinobacteria. Such variations might influence the catabolic activities or substrate utilization patterns of catechol 1,2-dioxygenase among Gordonia.