Wide-spectrum Amidase Research Articles

Cloning, sequence analysis and expression of the gene encoding a novel wide-spectrum amidase belonging to the amidase signature superfamily from Achromobacter xylosoxidans

Amidases are very important enzymes for industrial biocatalysis. We scored a novel amidase by screening the Achromobacter xylosoxidans gene library with cephalosporin analogous amides. The gene coding for the enzyme, designated ana, was cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis of ana showed it to be an amidase signature family member. Interestingly, we noted that almost all Ana homologous amidases are from human pathogens responsible for chronic lung infections. Knowing the genetic context of Ana and its homologous amidases, we suggest that they could be a part of transposon structure. Ana can efficiently hydrolyze a series of cephalosporin analogous amides, including amides with an aninine, p-nitro-aninine, and β-naphthylamine moiety, while cephalosporin could not serve as its substrate.

Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherichia coli and marked enhancement of amidase expression using directed evolution☆

A 1.6-kb DraI– HindIII DNA fragment from Bacillus stearothermophilus BR388 chromosomal DNA encoding a wide-spectrum amidase was cloned into Escherichia coli DH5α. With acrylamide substrate, the amidase showed maximum activity at 55°C, pH 7.0, and 0.12-M substrate, and demonstrated significant activity in 1-M acrylamide. A mutant prepared by PCR-based random mutagenesis of a 1.65 kb segment of B. stearothermophilus BR388 chromosomal DNA containing the amidase gene had two adenine bases replaced with guanine, resulting in a single primary structure alteration of His26 into Arg. This mutant demonstrated a 23-fold increase in amidase activity compared to wild–type, which is attributed to increased amidase gene transcription.

Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis

The study of acyl transfer activity of a wide spectrum amidase from Rhodococcus sp. R312, overproduced in an Escherichia coli strain, revealed that the ‘bi-bi-ping-pong’ type reaction was efficient with only four very-short chain (C 2–C 3) aliphatic amides as substrates. The optimum working pH was 7.0 for all neutral amides. Very short-chain aliphatic carboxylic acids were 10–1000-fold less efficient and the corresponding optimum working pH values depended on the acid used. Very polar molecules, such as water, hydroxylamine and hydrazine, were good acyl acceptors. An [acyl donor]/[acyl acceptor] ratio lower than 0.3-0.5 had to be maintained to avoid enzyme inhibition by excess acyl donor. The different acyl-enzyme complexes generally exhibited high affinity for hydroxylamine or hydrazine (except the propionyl-enzyme complex), so that the residual hydrolysis activities were almost totally inhibited at appropriate acyl acceptor concentrations. Molar conversion yields were higher with hydrazine as acyl acceptor (e.g., 97% with acetamide as acyl donor) because of the higher V max values, but in all cases, interesting quantities of short-chain hydroxamic acids (2.9-6.5 g l −1) and acid hydrazides (6.4–7.8 g l −1) could be quickly obtained (10–60 min) with small amounts of enzyme (0.04-0.20 g l −1).

The biotransformation of t-butylacetonitrile and its boron-containing analogue trimethylamine-cyanoborane by Brevibacterium R312.

The ability of the nitrile hydratase/amidase system from Brevibacterium R312 to biotransform tert-butylacetonitrile was studied with a view to their utilisation in the production of novel amino acids from isostructural compounds. Brevibacterium R312 was able to transform nitriles with this structure; however, the wide spectrum amidase from this organism was unable to biotransform the corresponding amide to the carboxylic acid.

Biocatalyst improvement for the production of short-chain hydroxamic acids

The wide spectrum amidase gene ( amiE) derived from Rhodococcus sp. R312 was overexpressed in a recombinant Escherichia coli strain. Amidase was extracted during the stationary phase by cell grinding. After partial purification with Q-Sepharose chromatography, the enzyme was immobilized on Duolite A-378 resin. A higher immobilization yield ( R i = 87%) was achieved with NaH 2 PO 4 Na 2 HPO 4 phosphate buffer (100 m m) at pH 7. Study of the immobilized amidase demonstrated that the physicochemical properties were similar to those of the free enzyme with respect to pH and activation energy. On the other hand, the optimal working temperature was higher for the immobilized amidase and thermostability was slightly improved. Ten g of insoluble biocatalyst produced 1 l of acetohydroxamic acid (at least 4.1 g l −1) solution after 90 min reaction time. The acetamide bioconversion rate was 55–60% (mol mol −1). After lyophilization, a 10 g powder containing 40% (wt wt −1) acetohydroxamic acid was recovered.

Characterization and cloning of an enantioselective amidase from Comamonas acidovorans KPO-2771-4

The characteristics and amino acid sequence of an enantiomer-selective amidase active on R-(−)-2-(3′-benzoyl phenyl)propionamide [ R-(−)-ketoprofen amide] purified previously from Comamonas acidovorans KPO-2771-4 were studied. On gel filtration, this amidase appeared to be a monomer with a molecular mass of 55 kDa. It had maximal activity at 35°C and at pH values from 8.5 to 10.0. Except for Cu 2+, Zn 2+, Pb 2+, and p-chloromercuribenzoate, it was not affected by chelating reagents, carbonyl reagents, reductants, most metal ions, or thiol reagents. The amidase had hydrolyzing activity against a broad range of aliphatic, aromatic, and amino acid amides, evidence that it is a wide-spectrum amidase. Oligonucleotide probes designed from limited peptide sequence information were used to clone the corresponding gene. The nucleotide-determined sequence indicated that the amidase consists of 473 amino acids ( M w 50,464 Da). Significant homologies were found at the amino acid level between the R-enantiomer-selective amidase of C. acidovorans KPO-2771-4 and the S-enantiomer-selective enzymes from Rhodococcus sp., Brevibacterium sp., and Pseudomonas sp. Only 39% homology was conserved in the consensus region of these amidases.

Sizing of theRhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation

The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) genome by pulsed-field gel electrophoresis. AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments.

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli.

The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli.

Optimization of culture conditions of Brevibacterium sp. for the production of amidase and adipamidase

The culture conditions required for the maximum production of the wide spectrum amidase and the adipamidase were determined for two strains of Brevibacterium sp. applying two mathematical techniques: the Hadamard matrix and the analyses of response surface. A culture medium was optimized and the role of Zn 2+ on the activity of adipamidase was demonstrated. The enzyme was shown to be not subject to catabolic repression by glucose or ammonium sulfat and it was not inducible. The influence of physical and chemical factors (pH, temperature) on cell growth and enzyme activity was also studied.

Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa

A Brevibacterium sp. R312 DNA fragment encoding the wide-spectrum amidase (EC 3.5.1.4) has been cloned and sequenced, using limited amino acid (aa) sequence information obtained from the purified enzyme. The deduced aa sequence showed more than 80% strict identity with the Pseudomonas aeruginosa aliphatic amidase, the product of the amiE gene, suggesting a horizontal transfer of the gene during evolution between Gram + and Gram − bacteria.

Purification and properties of the β‐glucosidase from a nitrile hydratase‐producing Brevibacterium sp. strain R312

Besides its nitrile hydratase and wide spectrum amidase activities, the Brevibacterium sp. R312 strain also possesses a constitutive beta-glucosidase. Its optimum pH is 6. The enzyme was purified by fractionation precipitation with ammonium sulfate followed by chromatographic elutions on Q-Sepharose Fast Flow, Sephadex G-200 and Phenyl Superose. The resulting purification was 1960 folds for a 6% yield. The molecular weight of this enzyme was estimated at 180,000. It contains two identical sub-units. The pI is 5.5. This enzyme has a strong affinity for aryl-beta-glycosides:pNPG, prunassine; it could also degrade linamarine. It is inhibited by p-chloromercuribenzoate, delta-gluconolactone and glucose.

The amidases from a Brevibacterium strain: study and applications.

In the first part of this paper different microbial enzyme systems able to hydrolyze the true amide function are reviewed, then some enzyme systems are described in detail: aminopeptidases, amidases of Aspergillus nidulans and of Pseudomonas aeruginosa.Following consideration of analytical techniques (NMR, GLC, TLC, colorimetric assay of the ammonium ion) useful in the study of amidases, the second part of the paper deals with the amidases of Brevibacterium sp. R312 and their applications. Selection conditions of the strain, demonstrations of the different amidase systems, and complete studies of the L-α-aminoamidase and the wide spectrum amidase are described. Some examples of applications and future developments are also given.

Acyltransferase Activity of the Wide Spectrum Amidase of Brevibacterium sp. R312

Summary: The wide spectrum amidase from Brevibacterium sp. R312, which can hydrolyse many amides tothe corresponding acids, was shown to transfer the acyl groups of amides, acids and esters to hydroxylamine. The transfer rates ofthese reactions in cytoplasmic fractions were measured and compared. The K m and V max were determined for different substrates in the presence of hydroxylamine. The enzyme was also shown to transfer the acyl group of the amide analogue N–methylacetamide to hydroxylamide and that of acetamide to the hydroxylamine analogue methylhydroxylamine.

Regulation of nitrile-hydratase synthesis in a Brevibacterium species.

The regulation of nitrile-hydratase biosynthese was studied in Brevibacterium sp R 312. Enzyme biosynthesis was not influenced by any carbon and nitrogen source used in the growth medium. It was, however, repressed by amide and amide analogues. Acetamide repressed nitrile-hydratase biosynthesis and induced the wide-spectrum amidase. Therefore, it appeared reasonable to hypothesize a single repressor gene for the nitrile-hydratase/wide-spectrum amidase system.