Diversity of Nitrile Hydratase and Amidase Enzyme Genes in Rhodococcus erythropolis Recovered from Geographically Distinct Habitats

Abstract

A molecular screening approach was developed in order to amplify the genomic region that codes for the ␣-and ␤-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis. A hydratase PCR product was also obtained from R. erythropolis DSM 43066 T , which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R. erythropolis strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R. erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel R. erythropolis enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R. erythropolis. Our findings indicate that the NHase and amidase genes present in geographically distinct R. erythropolis strains are not globally mixed.Nitrile hydratase (NHase) is a soluble bacterial metalloenzyme (41) that catalyzes the hydration of nitrile compounds to the corresponding amides, which may be converted by an amidase to the corresponding acids plus ammonia (40). NHase consists of two subunits (␣ and ␤), each with a molecular mass of approximately 23 kDa; the amino acid sequences of the subunits are not related, and the structural genes are normally adjacent in the same operon, although the ␣-and ␤-coding sequence order is variable (42). NHase is classified into two groups on the basis of the metal ion composing the catalytic center; an Fe-type NHase has a nonheme iron atom (74), and a Co-type NHase has a noncorrinoid cobalt atom (12) at the catalytic center. The Fe-and Co-type NHase enzymes differ in biotransformation activity and substrate specificity, although their amino acid sequences exhibit significant homology.NHase enzymes have considerable practical importance as biocatalysts for the industrial production of acrylamide and nicotinamide (84) and also for environmental bioremediation, where they have been shown to be effective in the removal of nitrile...

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier

“…With the ability to hydrolyze amides to corresponding optical pure compounds with high enantioselectivity, mild reaction conditions and environmental compatibility, amidases (EC 3.5.1.4) have been intensively studied [2,4,7,8,10,12,13,16,17,21,22,26,30]. However, current research on amidase, such as screening, characterization and its heterogeneous expression, is concentrated on the laboratory level, and applications of amidase on a large scale are quite few.…”

Characterization and Evaluation of the Potential of a Diesel-Degrading Bacterial Consortium Isolated from Fresh Mangrove Sediment

“…Rhodococcus are aerobic, Gram-positive, non-motile, nocardioform actinomycetes, with a life cycle alternating between cocci and small rods, sometimes showing small filamentous projections (Bicca et al, 1999). R. erythropolis has the ability to occupy many niches; this species dominates the nitrile-degrading microorganisms from many marine and terrestrial zones (Brandao et al, 2003). Diesel oil is an excellent model for studying hydrocarbon biodegradation, since it is constituted of a variety of these molecules, such as paraffin, olefins, naphtha, and aromatic compounds.…”

Nitrile-Degrading Bacteria Isolated from Compost

“…The deduced protein sequences showed highest identity to the alpha subunit of Rhodococcus sp. N-774 (Ikehata et al, 1989) and the beta subunit of R. erythropolis deepsea strain 122-AN065 (Brandão et al, 2003). The deduced acetamidases (BKP42_40660 and BKP42_26750) are most similar to acetamidases of R. erythropolis and Rhodococcus sp.…”

“…Similarity searches for putative genes involved in nitrile-degradation revealed two genes (BKP42_65670 and BKP42_65680), coding for one of the two putative NHase subunits. These enzymes are known to be responsible for nitrile degradation in various R. erythropolis strains (Kaufmann et al, 1999;Brandão et al, 2003;Vejvoda et al, 2007;Kamble et al, 2013). The deduced protein sequences showed highest identity to the alpha subunit of Rhodococcus sp.…”