Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans.

A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.     

Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp. denitrificans PN-1

Pseudomonas sp. strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp. denitrificans PN-1. Strain PN-1 is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does not ferment sugars. Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4 kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa and Pseudomonas stutzeri strains. A restriction endonuclease map was constructed.     

Thiodiglycol Metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C–S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO₄ ^2– ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.     

Thiodiglycol metabolism in Alcaligenes xylosoxydans subsp. denitrificans

The investigation of the degradation of thiodiglycol (the major product of mustard gas hydrolysis) by Alcaligenes xylosoxydans subsp. denitrificans strain TD2 showed that thiodiglycol is metabolized through the oxidation of its primary alcohol groups and the subsequent cleavage of C-S bonds in the intermediate products, thiodiglycolic and thioglycolic acids. The end products of these reactions are SO4(2-) ions and acetate, the latter being involved in the central metabolism of strain TD2. The oxidation of the sulfur atom gives rise to diglycolsulfoxide, which is recalcitrant to further microbial degradation. Based on the data obtained, a metabolic pathway of thiodiglycol transformation by A. xylosoxydans subsp. denitrificans strain TD2 is proposed.     

Production of d-Aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4

A bacterial strain that produces d-aminoacylase was isolated from soil and identified as Alcaligenes denitrificans subsp. xylosoxydans MI-4. l-Aminoacylase activity in this strain was only 1 to 2% of d-aminoacylase activity. d-Aminoacylase was inducibly produced. N-Acetyl-dl-leucine was the best inducer, and the d-isomer had the ability to induce the enzyme. Enzymatic resolution of N-acetyl-dl-methionine with the crude enzyme was carried out, and the d/l ratio in the resolved methionine was approximately 100/7, suggesting that resolution with crude enzymes may become possible by removing small amounts of the contaminated l-form with l-amino acid oxidase.     

Effect of microbial competition on the survival and activity of 2,4-D-degrading Alcaligenes xylosoxidans subsp. denitrificans added to soil

In laboratory experiments samples of natural or chloroform-fumigated soils were inoculated with an Alcaligenes xylosoxidans subsp. denitrificans which is able to use 2,4-dichlorophenoxyacetic acid (2,4-D) as a sole carbon source. Biotic factors affecting survival and activity of the inoculant were determined. In natural soil the numbers and activity of Alc. xylosoxidans declines in few days. The strain proliferated only when it was inoculated immediately after soil fumigation. Its activity 15 d after inoculation was then twice its initial activity. When inoculation of fumigated samples was delayed, the numbers of Alc. xylosoxidans declined, but its activity was higher than in the natural soil. Addition of soil bacteria or fungi resulted in a reduction in the numbers and activity of Alc. xylosoxidans. These results suggest that microbial competition for nutrients and biological spaces causes the decline in the population and activity of inoculant added to soil.     

Cloning,expression and characterization of d-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173

d-Aminoacylase catalyzes the conversion of N-acyl-d-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the d-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized d-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-d-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized d-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS–PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and 50 °C, and was stable at pH 6.0–8.0 and up to 45 °C. Its activity was inhibited by Cu²⁺, Fe²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Zn²⁺ and Hg²⁺, whereas Mg²⁺ had no significant influence on this recombinant d-aminoacylase. This is the first report on the characterization of d-aminoacylase with activity towards both N-acyl derivatives of neutral d-amino acids and N-acyl-d-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of d-amino acids.     

Cloning and nucleotide sequence of a D,L-haloalkanoic acid dehalogenase encoding gene from Alcaligenes xylosoxidans ssp. denitrificans ABIV

We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L-isomer specific dehI dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a ⁵⁴ activator protein, similar to the dehR _Iregulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D-or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.     

Characterization of D-aminoacylase from Alcaligenes denitrificans DA181.

The D-aminoacylase produced by Alcaligenes denitrificans DA181 was a new type of aminoacylase which had both high stereospecificity and specific activity. The molecular weight and isoelectric point of this enzyme were 58,000 and 4.4, respectively. The apparent Km and kcat values of this enzyme for N-acetyl-D-methionine were estimated to be 0.48 mM and 6.24 x 10(4) min-1, respectively. The optimum temperature was 45 degrees C. The enzyme was stable up to 55 degrees C for 1 hr in the presence of 0.2 mg/ml bovine serum albumin. The enzyme was stable in the pH range of 6.0 to 11.0 with an optimum pH of 7.5. This enzyme contained about 2.1 g atom of zinc per mole of enzyme. Enzyme activity was inhibited by incubation with EDTA. The inhibition by EDTA was fully reversed by Co2+ and partially by Zn2+.     

Sarcosine Oxidase Involved in Creatinine Degradation in Alcaligenes denitrificans subsp. denitrificans J9 and Arthrobacter spp. J5 and J11

Three microorganisms that degrade creatinine and contain sarcosine oxidase were isolated from soil and identified to be Alcaligenes denitrificans subsp. denitrificans J9 and Arthrobacter spp. J5 and J11. The three soil isolates degraded creatinine only via creatine by inducibly formed creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase when cultivated with creatinine as the main nitrogen source. Sarcosine dehydrogenase, creatinine deiminase, and N-carbamoylsarcosine amidohydrolase were not induced by creatinine. Other microorganisms that degrade creatinine all contain sarcosine dehydrogenase as the enzyme for sarcosine oxidation, so these isolates seem to be unique in having sarcosine oxidase involved in their processes of creatinine degradation. Sarcosine oxidase was purified from A. denitrificans subsp. denitrificans J9 and partially characterized.     

Analysis of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pEST4011 of Achromobacter xylosoxidans subsp. denitrificans strain EST4002

The 2,4-dichlorophenoxyacetic acid (2,4-D)-degradative bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002, isolated in Estonia more than 10years ago, was found to contain the 70kb plasmid pEST4011 that is responsible for the bacterium having had obtained a stable 2,4-D(+) phenotype. The tfd-like genes for 2, 4-D degradation of the strain EST4002 were located on a 10.5kb region of pEST4011, but without functional genes coding for chloromuconate cycloisomerase and chlorodienelactone hydrolase. The latter two genes are probably encoded by homologous, tcb-like genes, located elsewhere on pEST4011. We also present evidence of two copies of insertion element IS1071-like sequences on pEST4011. IS1071 is a class II (Tn3 family) insertion element, associated with different catabolic genes and operons and globally distributed in the recent past. We speculate that this insertion element might have had a role in the formation of plasmid pEST4011. The 28kb plasmid pEST4012 is generated by deletion from pEST4011 when cells of A. xylosoxidans EST4002 are grown in the absence of 2,4-D in growth medium. We propose that this is the result of homologous recombination between the two putative copies of IS1071-like sequences on pEST4011.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Characterization of D-Aminoacylase from Alcaligenes denitrificans DA181

The D-aminoacylase produced by Alcaligenes denitrificans DA181 was a new type of aminoacylase which had both high stereospecificity and specific activity. The molecular weight and isoelectric point of this enzyme were 58,000 and 4.4, respectively. The apparent K_m and k_cat values of this enzyme for N-acetyl-D-methionine were estimated to be 0.48 him and 6.24 × 10⁴ min respectively. The optimum temperature was 45°C. The enzyme was stable up to 55°C for 1 hr in the presence of 0.2 mg/ml bovine serum albumin. The enzyme was stable in the pH range of 6.0 to 11.0 with an optimum pH of 7.5. This enzyme contained about 2.1 g atom of zinc per mole of enzyme. Enzyme activity was inhibited by incubation with EDTA. The inhibition by EDTA was fully reversed by Co²⁺ and partially by Zn²⁺.     

Purification and characterization of aromatic amine dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on beta-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (alpha2beta2) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the alpha-subunit (42.3-kDa subunit) and the beta-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the beta-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and beta-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 degrees C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, beta-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 microM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 degrees C for one month at least in phosphate buffer (pH 7.0).     

Purification and Characterization of Aromatic Amine Dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on β-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (α₂β₂) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the α-subunit (42.3-kDa subunit) and the β-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the β-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and β-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 °C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, β-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 μM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 °C for one month at least in phosphate buffer (pH 7.0).     

Purification and preliminary crystallographic studies on azurin and cytochrome c' from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015.

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C222₁ (with marked tetragonal pseudo-symmetry), cell dimensions $\text{a = 75.0}\text{A}\text{?}\text{, b = 74.1}\text{A}\text{?}\text{, c = 99.5}\text{A}\text{?}$, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6₁22 (or P6₅22), cell dimensions $\text{a = b = 54.7}\text{A}\text{?}\text{, c ~ 185}\text{A}\text{?}$, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.     

Purification and Characterization of l-Aminoacylase from Alcaligenes denitrificans DA181

The l-aminoacylase produced intracellularly by Alcaligenes denitrificans DA181 was puritied to homogeneity. This enzyme had an apparent molecular weight of 80,000, and was composed of two subunits of identical molecular weight. Its isoelectric point was pH 5.1. The optimal reaction temperature and pH were 65°C and 8.0, respectively. This enzyme showed specificity toward N-acetyl-derivative of hydrophobic l-amino acids with N-acetyl-l-valine as the favored substrate, followed by N-acetyl-l-alanine.