Screening, Isolation and Taxonomical Properties of Microorganisms Having Acrylonitrile-hydrating Activity

Watanabe, Ichirô; Satoh, Yoshiaki; Enomoto, Kanehiko

doi:10.1080/00021369.1987.10868585

Cited by 19 publications

(23 citation statements)

References 4 publications

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“…Therefore, even though geographically distinct environmental strains may have identical NHase gene sequences, it does not follow that their activity profiles are identical. Similar findings were obtained for Rhodococcus strains N-771, N-774, and AJ270, isolated from different soil samples, which although possessing identical NHase gene and amino acid sequences, had distinct nitrile-transforming activities (6,81).…”

Section: Discussionsupporting

confidence: 70%

“…However, despite this biotechnological relevance, information concerning the distribution of nitrile-hydrolyzing enzymes in microorganisms and in the environment is quite limited. Bacteria having NHase enzymes have been recovered from shallow marine sediment (45), deepsea sediments (18, 31), geothermal habitats (65, 85), and various soils (21,46,47,48,81), including contaminated soils (6). Recently, we reported the widespread distribution of nitrilehydrolyzing actinomycetes in marine sediments and terrestrial soils (9).…”

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confidence: 99%

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

Brandão

Clapp

Bull

2003

Appl Environ Microbiol

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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...

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Section: Discussionsupporting

confidence: 70%

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confidence: 99%

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

Brandão

Clapp

Bull

2003

Appl Environ Microbiol

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“…The identity at the DNA level between the sequences of such long fragments from the two strains strongly argues in favor of Rhodococcus sp. strain N-774 (28) and Brevibacterium sp. strain R312 (1) being identical, or at least very closely related, microorganisms.…”

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confidence: 99%

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase

Mayaux¹,

Cerebelaud²,

Soubrier³

et al. 1990

J Bacteriol

151

118

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An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 imol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30TC under the control of an E. coli promoter and ribosome-binding site.

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“…The alternative pathway involves nitrile hydratases which mediate the conversion of nitriles to the corresponding amides. In a subsequent step amidases convert the amides to the corresponding carboxylic acids and ammonia (Harper, 1977a,b;Nagasawa et al, 1987Nagasawa et al, , 1988aNagasawa et al, ,b, 1990Watanabe et al, 1987). The enzymatic hydrolysis of nitriles represents a very convenient synthetic method for amides and/or carboxylic acids due to the mild reaction conditions.…”

Section: Discussionmentioning

confidence: 99%

Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates

Layh

Stolz

Böhme

et al. 1994

Journal of Biotechnology

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Bacteria were enriched from soil samples with succinate as a carbon source and racemic naproxen nitrile [2-(6-methoxy-2-naphthyl)propionitrile] as sole source of nitrogen. Since naproxen nitrile was only poorly soluble in water media amended with different water-immiscible organic phases were used for the enrichments. With pristane (2,6,10,14-tetramethylpentadecane) as the organic phase two bacterial strains were isolated (strain C3II and strain MP50) which were identified as rhodococci. Cells of both strains converted naproxen nitrile via naproxen amide to naproxen. From racemic naproxen nitrile Rhodococcus sp. C3II formed S-naproxen amide and subsequently S-naproxen. Racemic naproxen amide was hydrolysed to S-naproxen. Rhodococcus sp. MP50 converted racemic naproxen nitrile predominantly to R-naproxen amide and racemic naproxen amide to S-naproxen. With both strains racemic naproxen amide was converted to S-naproxen with an enantiomeric excess > 99% at a conversion rate up to 80% of the theoretical value. In strain C3II the enzymes which hydrolysed naproxen nitrile and naproxen amide were present only at a low constitutive level. In contrast, in Rhodococcus sp. MP50 these activities were induced when grown in the presence of various nitriles.

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Screening, Isolation and Taxonomical Properties of Microorganisms Having Acrylonitrile-hydrating Activity

Cited by 19 publications

References 4 publications

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

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

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase

Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates

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