• Journal of Microbiology
• /
• v.35 no.4
• /
• pp.295-299
• /
• 1997

Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were $30{\sim}35^{\circ}C$ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

• BMB Reports
• /
• v.34 no.2
• /
• pp.172-175
• /
• 2001

Catechol 1,2-dioxygenase $I_1$ ($CDI_1$) is the first enzyme of the $\beta$-ketoadipate pathway in Acinetobacter lowffii K24. $CDI_1$ has two cysteines (155, 202) and its enzyme activity is inhibited by the cysteine inhibitor, $AgNO_3$. Two mutants, $CDI_1$ C155V and $CDI_1$ C202V, were obtained by site-directed mutagenesis. The two mutants were overexpressed and the mutated amino acid residues (Cys$\rightarrow$Val) were characterized by peptide mapping and amino acid sequencing. Interestingly, $CDI_1$ C155V was inhibited by $AgNO_3$, whereas $CDI_1$ C202V was not inhibited. This suggests that $Cys^{202}$ is the sole inhibition site by $AgNO_3$ and is close to the active site of the enzyme. However, the results of the biochemical assay of mutated $CDI_1s$ suggest that the two cysteines are not directly involved in the activity of the catechol 1,2-dioxygenase of $CDI_1$.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.12
• /
• pp.1995-1999
• /
• 2006

Pseudomonas sp. K82 cultured in p-hydroxybenzoate induces protocatechuate 4,5-dioxygenase (PCD 4,5) for p-hydroxybenzoate degradation. In this study, a 6.0-kbp EcoR1 fragment containing p-hydroxybenzoate degradation genes was cloned from the genome of Pseudomonas sp. K82. Sequence analysis identified four genes, namely, pcaD, pcaA, pcaB, and pcaC genes known to be involved in p-hydroxybenzoate degradation. Two putative 4-hydroxyphenylpyruvate dioxygenases and one putative oxidoreductase were closely located by the p-hydroxybenzoate degradation genes. The gene arrangement and sequences of these p-hydroxybenzoate degradation genes were similar to those of Comamonas testosteroni and Pseudomonas ochraceae. PcaAB (PCD4,5) was overexpressed in the expression vector pGEX-4T-3, purified using a GST column, and confirmed to have protocatechuate 4,5-dioxygenase activity. The N-terminal amino acid sequences of overexpressed PCD4,5 were identical with those of purified PCD4,5 from Pseudomonas sp. K82.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.1
• /
• pp.35-42
• /
• 2000

Reaction characteristics of 4-methylcatechol 2,3-dioxygenase (4MC230) purified from Pseudomonas putida SU10 with a higher activity toward 4-methylcatechol than catechol or 3-cethylcatechol were studied by altering their physical and chemical properties. The enzyme exhibited a maximum activity at pH 7.5 and approximately 40% at pH 6.0 for 4-methylcatechol hydrolysis. The optimum temperature for the enzyme was around $35^{\circ}C$, since the enzyme was unstable at higher temperature. Acetone(10%) stabilized the 4MC230. The effects of solvent and other chemicals (inactivator or reactivator) for the reactivation of the 4MC230 were also investigated. Silver nitrate and hydrogen peroxid severely deactivated the enzyme and the deactivation by hydrogen peroxide severely deactivated the enzyme and the deactivation by hydrogen peroxide was mainly due to the oxidation of ferrous ion to ferric ion. Some solvents acted as an activator and protector for the enzyme from deactivation by hydrogen peroxide. Ascorbate, cysteine, or ferrous ion reactivated the deactivated enzyme by hydrogen peroxide. The addition of ferrous ion together with a reducing agent fully recovered the enzyme activity and increased its activity abut 2 times.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.4
• /
• pp.475-482
• /
• 2014

The metabolic pathway of eugenol degradation by thermophilic Geobacillus sp. AY 946034 strain was analyzed based on the lack of data about eugenol degradation by thermophiles. TLC, GC-MS, and biotransformation with resting cells showed that eugenol was oxidized through coniferyl alcohol, and ferulic and vanillic acids to protocatechuic acid before the aromatic ring was cleaved. The cell-free extract of Geobacillus sp. AY 946034 strain grown on eugenol showed a high activity of eugenol hydroxylase, feruloyl-CoA synthetase, vanillate-O-demethylase, and protocatechuate 3,4-dioxygenase. The key enzyme, protocatechuate 3,4-dioxygenase, which plays a crucial role in the degradation of various aromatic compounds, was purified 135-fold to homogeneity with a 34% overall recovery from Geobacillus sp. AY 946034. The relative molecular mass of the native enzyme was about $450{\pm}10$ kDa and was composed of the non-identical subunits. The pH and temperature optima for enzyme activity were 8 and $60^{\circ}C$, respectively. The half-life of protocatechuate 3,4-dioxygenase at the optimum temperature was 50 min.

• Korean Journal of Microbiology
• /
• v.55 no.1
• /
• pp.55-57
• /
• 2019

The Runella sp. ABRDSP2, capable of degrading mono-aromatic compounds such as toluene, was isolated from freshwater. The whole genome, consisting of a circular single chromosome and three plasmids, was composed of total 7,613,819 bp length with 44.4% G+C contents and 6,006 genes. The genome of strain ABRDSP2 contains many aromatic hydrocarbon degrading genes such as monooxygenase, ring-cleaving dioxygenase, and catechol 1,2-dioxygenase. The complete genome reveals versatile biodegradation capabilities of Runella sp. ABRDSP2.

• Microbiology and Biotechnology Letters
• /
• v.26 no.4
• /
• pp.352-357
• /
• 1998

Biodegradation of benzoate and m-toluate was investigated using a Pseudomonas sp. isolated in a continuous culture for 45 days with a step-wise increase of the subsrates. The optimum mixture ratio of benzoate and m-toluate was 75% and 25%, respectively. During 45-day culture, removal of benzoate and m-toluate, which was replaced 2,000 ppm on the 30th day were 94% and 79%, respectively, when COD removal rate was 80%. The enzymatic activity of catechol 1,2-dioxygenase increased and that of catechol 2,3-dioxygenase decreased as the concentration of m-toluate was increased. These results suggested that m-toluate induced enzyme activity for degradation of benzoate. The shape of isolated strain in the continuous culture was investigated with SEM and the results showed that the cell shape was more damage according to the higher concentration of aromatic hydrocarbons. Therefore, we suggested that the tolerance against aromatic hydrocarbons was related to not only enzymatic activity but also characteristic of cell membrane or cell wall.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2001.11a
• /
• pp.16-25
• /
• 2001

A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.10
• /
• pp.1440-1445
• /
• 2010

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 were 8.0, $30^{\circ}C$, and 175 rpm, respectively. In addition, low concentrations of glucose were found to inhibit the degradation of DBP, whereas high concentrations of glucose increased its degradation. Meanwhile, a substrate utilization test showed that JDC-11 was also able to utilize other phthalates. The major metabolites of DBP degradation were identified as monobutyl phthalate and phthalic acid by gas chromatography-mass spectrometry, allowing speculation on the tentative metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11. Using a set of new degenerate primers, a partial sequence of the 3,4-phthalate dioxygenase gene was obtained from JDC-11. Moreover, a sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of the phthalate dioxygenase from Rhodococcus coprophilus strain G9.

• Journal of Microbiology
• /
• v.40 no.1
• /
• pp.38-42
• /
• 2002

The strain KKI isolated from soil contaminated with polycyclic aromatic hydrocarbons was identified as Pseundomonas sp. based on analyses by MIDI and Biolog Identification System. Cellular and physiological responses of strain KKI to two-ring polycyclic aromatic hydrocarbon, naphthalene were evaluated using radiorespirometry, PLFAs and sequence analysis of Rieske-type iron sulfur center of dioxygenase. KKI was found to be able to rapidly mineralize naphthalene. Notably, KKI cells pregrown on phenanthrene were able to mineralize naphthalene much more rapidly than naphthalenepregrown cells. The total cellular fatty acids of KKI were comprised of eleven C-even and two C-odd fatty acids (fatty acids < 0.2% in abundance were not considered in this calculation). Lipids 12:0 2OH, 12:03 OH, 16:0, 18:1 6c, 18:0 increased for naphthalene-exposed cells, while lipids 18:1 7c1/15:0 ism 2OH, 17:0 cyclo, 18:1 7c, 19:0 cyclo decreased. Data from Northern hybridization using a naphthalene dixoygenase gene fragment cloned out from KKI as a probe provided the information that naphthalene dioxygenase gene was more highly expressed in cells grown on phenanthrene than naphthalene.