scholarly journals Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein

1992 ◽  
Vol 285 (2) ◽  
pp. 661-666 ◽  
Author(s):  
M C Silvestrini ◽  
F Cutruzzolà ◽  
R D'Alessandro ◽  
M Brunori ◽  
N Fochesato ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Recombinant Protein ◽  
Pseudomonas Putida ◽  
Molecular Mass ◽  
Nitrite Reductase ◽  
Water Soluble ◽  
Binding Kinetics ◽  
Cytochrome C551

Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).

Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82

1999 ◽  
Vol 87 (6) ◽  
pp. 721-731 ◽  
Author(s):  
Noboru Takizawa ◽  
Toshiya Iida ◽  
Takashi Sawada ◽  
Kazuhiro Yamauchi ◽  
Yue-Wu Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pseudomonas Putida ◽  
Nucleotide Sequences ◽  
Genes Encoding

Purification and Characterization of Gentisate 1,2-Dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869

1999 ◽  
Vol 65 (3) ◽  
pp. 946-950 ◽  
Author(s):  
Yongmei Feng ◽  
Hoon Eng Khoo ◽  
Chit Laa Poh
Keyword(s):  
Pseudomonas Putida ◽  
Molecular Mass ◽  
Amino Acid Residues ◽  
Stability Range ◽  
Native Molecular Mass ◽  
Heat Stable ◽  
A Subunit ◽  
Pseudomonas Alcaligenes ◽  
Similar Kinetic

ABSTRACT Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1,2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1,2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Km s of 92 and 143 μM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with k cat/Km values of 44.08 × 104 s−1 M−1 for the P25X enzyme and 39.34 × 104 s−1M−1 for the P35X enzyme. Higherk cat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1,2-dioxygenases was around 50°C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.

Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11

2006 ◽  
Vol 52 (2) ◽  
pp. 157-168 ◽  
Author(s):  
Subhas Das ◽  
Dileep Kumar Singh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Half Life ◽  
Indoleacetic Acid ◽  
Bacterial Strains ◽  
Purification And Characterization ◽  
Temperature Optimum ◽  
Microbial Biodegradation ◽  
Membrane Bound

A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophos-degrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34–41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%–16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect.Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11.

Characterization of saccharolytic nonfermentative bacteria associated with man

1970 ◽  
Vol 16 (5) ◽  
pp. 351-362 ◽  
Author(s):  
M. J. Pickett ◽  
Margaret M. Pedersen
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Pseudomonas Putida ◽  
Pseudomonas Fluorescens ◽  
Pseudomonas Stutzeri ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Clinical Strains ◽  
Pseudomonas Maltophilia ◽  
Reference Strains

Features of 378 clinical isolates of saccharolytic, nonfermentative Gram-negative rods and 20 reference strains were examined. All but four of the clinical strains were assigned to recognized taxa, namely Acinetobacter, Chromobacterium, Flavobacterium, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas maltophilia, Pseudomonas multivorans, Pseudomonas putida, Pseudomonas stutzeri, and Xanthomonas.

Isolation and characterization of the d1 domain of Pseudomonas aeruginosa nitrite reductase

1996 ◽  
Vol 62 (2) ◽  
pp. 77-87 ◽  
Author(s):  
Maria Chiara Silvestrini ◽  
Francesca Cutruzzolà ◽  
Maria Eugenia Schininà ◽  
Bruno Maras ◽  
Gabriella Rolli ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Isolation And Characterization

scholarly journals Intramolecular Electron Transfer in Pseudomonas aeruginosa cd1 Nitrite Reductase: Thermodynamics and Kinetics

2009 ◽  
Vol 96 (7) ◽  
pp. 2849-2856 ◽  
Author(s):  
Ole Farver ◽  
Maurizio Brunori ◽  
Francesca Cutruzzolà ◽  
Serena Rinaldo ◽  
Scot Wherland ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Nitrite Reductase ◽  
Intramolecular Electron Transfer ◽  
Thermodynamics And Kinetics

scholarly journals Eukaryotic nirK Genes Encoding Copper-Containing Nitrite Reductase: Originating from the Protomitochondrion?

2009 ◽  
Vol 75 (9) ◽  
pp. 2652-2658 ◽  
Author(s):  
Sang-Wan Kim ◽  
Shinya Fushinobu ◽  
Shengmin Zhou ◽  
Takayoshi Wakagi ◽  
Hirofumi Shoun
Keyword(s):  
Recombinant Protein ◽  
Phylogenetic Tree ◽  
Fusarium Oxysporum ◽  
Phylogenetic Relationship ◽  
Nitrite Reductase ◽  
Nitrate Respiration ◽  
Orthologous Genes ◽  
Genes Encoding ◽  
Genome Analyses

ABSTRACT Although denitrification or nitrate respiration has been found among a few eukaryotes, its phylogenetic relationship with the bacterial system remains unclear because orthologous genes involved in the bacterial denitrification system were not identified in these eukaryotes. In this study, we isolated a gene from the denitrifying fungus Fusarium oxysporum that is homologous to the bacterial nirK gene responsible for encoding copper-containing nitrite reductase (NirK). Characterization of the gene and its recombinant protein showed that the fungal nirK gene is the first eukaryotic ortholog of the bacterial counterpart involved in denitrification. Additionally, recent genome analyses have revealed the occurrence of nirK homologs in many fungi and protozoa, although the denitrifying activity of these eukaryotes has never been examined. These eukaryotic homolog genes, together with the fungal nirK gene of F. oxysporum, are grouped in the same branch of the phylogenetic tree as the nirK genes of bacteria, archaea, and eukaryotes, implying that eukaryotic nirK and its homologs evolved from a single ancestor (possibly the protomitochondrion). These results show that the fungal denitrifying system has the same origin as its bacterial counterpart.

Sign in / Sign up

Export Citation Format

Share Document