Ryu Shinke

Classification of catechol 1,2-dioxygenase family: sequence analysis of a gene for the catechol 1,2-dioxygenase showing high specificity for methylcatechols from Gram+ aniline-assimilating Rhodococcus erythropolis AN-13.

Gram+ aniline-assimilating Rhodococcus erythropolis AN-13 (AN-13) produces catechol 1,2-dioxygenase (C12O) showing high enzymatic activities for 3- and 4-methylcatechols [Aoki et al. (1984) Agric. Biol. Chem. 48, 2087-2095]. A 3.0 kb Sau3AI fragment carrying a gene encoding C12O(catA) was cloned by selection of transformants showing C12O activity from a gene library of AN-13. Furthermore, we specified a 1.6 kb SalI fragment containing catA from the Sau3AI fragment by subcloning. Sequence analysis revealed that the 1.6 kb SalI fragment carried a 855 bp open reading frame (ORF) encoding the entire AN-13 catA, preceded by a potential ribosome binding site (RBS). From comparison of the deduced amino acid (aa) sequence of C12O from AN-13 with other C12O reported previously, it was found that the AN-13 enzyme shares 56.0% aa sequence identity with C12o from Arthrobacter sp. mA3 (mA3) [Eck and Belter (1991) Gene 123, 87-92] compared with less than 36.4% aa sequence identities with others. In conclusion, we classified all C12O including the AN-13 enzyme into three subfamilies on the basis of similarity of aa sequences, numbers of aa residues, and substrate specificity.

Cloning and sequence analysis of two catechol-degrading gene clusters from the aniline-assimilating bacterium Frateuria species ANA-18

The aniline-assimilating bacterium Frateuria species ANA-18 produced two catechol 1,2-dioxygenases, CD I and CD II, and two muconate cycloisomerases, MC I and MC II. The catA genes catA1 and catA2 encoding CD I and CD II, respectively, were cloned from a gene library of this bacterium. The catA1 gene was clustered with catB1 encoding MC I, catC1 encoding muconolactone isomerase (MI), catD encoding beta-ketoadipate enol-lactone hydrolase (ELH), and ORFR1 encoding a putative LysR-type regulator. The organization of these genes was ORFR1catB1C1D. The catA2 gene also constructed a gene cluster involving catB2 encoding MC II, catC2 encoding MI, and ORFR2 encoding a putative LysR-type regulator with the alignment of ORFR2catB2A2C2. The intergenic regions of ORFR1-catB1 and ORFR2-catB2 contained homologous sequences with the catR-catB intergenic region containing a repression binding site and activation binding site of CatR in Pseudomonas putida. These findings suggest that the two cat clusters were regulated independently in their expression. When a product of cloned catD was added to a reaction mixture containing beta-ketoadipate enol-lactone, beta-ketoadipate was produced. This observation showed that the cloned catD encoded ELH and was expressed in Escherichia coli. We found that Frateuria sp. ANA-18 had a large plasmid with a molecular size more than 100kb. Polymerase chain reaction amplifying partial catA genes and Southern hybridization analyses with probes containing catA genes were conducted, to examine the localization of the two catA genes. We concluded that the catA1 and catA2 genes were located on the chromosomal and large plasmid DNAs, respectively, in Frateuria sp. ANA-18.

Metabolism of Aniline by Rhodococcus erythropolis AN–13

The metabolic pathway of aniline was examined in Rhodococcus erythropolis AN-13 that was isolated from soil when aniline was provided as a sole source of carbon and nitrogen. cis, cis-Muconic acid and β-ketoadipic acid were detected by thin-layer chromatography in an incubation mixture containing aniline and resting cells of this strain. These two carboxylic acids were also formed from catechol, when the substrate was incubated with cell-free extract of aniline-grown cells, and characterized spectrally as crystalline samples. Ammonia was released from aniline by resting cells. The cell-free extract of aniline-grown cells had a strong catechol 1,2-dioxygenase activity. Catechol, once formed from aniline, was apparently converted so rapidly to cis, cis-muconic acid that it could not be isolated. These results suggest that R. erythropolis AN-13 converted aniline to catechol with the release of ammonia and then mineralized catechol ultimately to inorganic end products, H2O and CO2, through the β ketoadipic ac...

Isolation of Aniline-assimilating Bacteria and Physiological Characterization of Aniline Biodegradation in Rhodococcus erythropolis AN-13

Twenty bacterial strains were isolated from soil, when aniline was provided as a sole source of carbon and nitrogen. Rhodococcus erythropolis AN-13, one of the isolates, grew on aniline at concentrations from 0.65 to 2.6mg/ml and completely degraded it. Ammonia was released from aniline with cell growth and accumulated in the cultural broth. Aniline-grown cells of this bacterium oxidized aniline and catechol, but did not take up a significant amount of oxygen in the presence of o- or p-aminophenol. These facts suggest that R. erythropolis AN-13 metabolized aniline through catechol. Additional carbon and nitrogen sources did not cause any repression of aniline biodegradation in this strain, but promoted its breakdown with an increase of cell growth. The bacterium used aniline in preference to glucose in a medium containing these two substrates.

Metabolism of 2-aminophenol by Pseudomonas sp. AP-3: modified meta-cleavage pathway

Abstract A novel pathway for 2-aminophenol metabolism by Pseudomonas sp. AP-3 is proposed. The proposed pathway is similar to that known for meta-cleavage of catechol except that one of the hydroxyl groups on the metabolites is replaced by an amino group. During the degradation of 2-aminophenol, 2-amino-2,4-pentadienoic acid is the last metabolite containing an amino group. We, therefore, propose a modified meta-cleavage pathway for the 2-aminophenol metabolism.

Partial purification and characterization of a bacterial dioxygenase that catalyzes the ring fission of 2-aminophenol

Abstract A bacterial strain AP-3, grown on 2-aminophenol as a sole carbon, nitrogen and energy source and isolated from soil, was identified as a Pseudomonas species. When Pseudomonas sp. AP-3 grew with this substrate, it synthesized an enzyme acting on 2-aminophenol. This enzyme was purified with an 103-fold increase of the specific activity from its cell-free extracts. We proved that the purified enzyme was a dioxygenase catalyzing the ring fission between 1- and 6-positions of 2-aminophenol with the consumption of 1 mol of O 2 per mol of substrate. The enzyme showed the maximal activity at pH 7.5. It was stable between pH 7.5 and 9 and up to 35°C. The molecular mass of this enzyme was 140 kDa by gel filtration.

Anaerobic synthesis of extracellular proteases by the soil bacterium Bacillus sp. AM-23: Purification and characterization of the enzymes

Abstract The anaerobic synthesis, purification and characterization of proteases from soil bacteria were investigated. We isolated a facultative anaerobe, Bacillus sp. AM-23, which synthesized extracellular proteases under anaerobic conditions. The bacterium generated two proteases (Protease I and II) on anaerobic culture, which were separated from each other by chromatography on DE-52 cellulose and purified to homogeneity by successive chromatographic techniques. The two proteases were similar to each other in molecular and catalytic properties. The molecular masses of Protease I and II were 20 kDa by gel filtration and 21 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two enzymes showed the maximal activity at pH 7.6 and were stable up to 55°C in the presence of Ca 2+ . Protease I and II were stable in the range of pH 6.0–8.2 and 6.5–8.5, respectively. Because diisopropyl fluorophosphate strongly inhibited the two proteases, the enzymes were presumed to be serine proteases. The two enzymes showed high hydrolytic activities for peptides with hydrophobic amino acid residues. The results revealed that there were several differences in molecular mass and substrate specificity between these “anaerobic” proteases and the serine proteases from aerobic microorganisms and animals.

A high level of accumulation of 2-hydroxymuconic 6-semialdehyde from aniline by the transpositional mutant Y-2 of Pseudomonas species AW-2

Four transpositional mutants of aniline-assimilating Pseudomonas sp. AW-2 produced 2-hydroxymuconic 6-semialdehyde (HMS) from aniline and accumulated it in a cultural medium. Among the four mutants, strain Y-2 produced the greatest amount of HMS (0.77 mg/ml) from 1 mg/ml of aniline hydrochloride in a 15-h growing culture. The conversion rate of aniline to HMS was 70% on a molar basis. Resting cells of strain Y-2 produced 0.65 mg/ml of HMS during 4h of incubation in a reaction mixture containing 1 mg/ml of aniline hydrochloride (conversion rate, 60%). Transposon Tn5-Mob was found to be inserted into the gene of HMS dehydrogenase in strain Y-2 by sequence analysis.

Capillary isotachophoretic determinations of metal ions by use of complexation equilibria in acetone-water medium

The determination of metal ions by capillary isotachophoresis and the complexation equilibria between metal ions and polyaminopolycarboxylic acids has been investigated. A seven-component mixture of metal ions can be separated in 45% v/v acetone-water medium when EDTA or DCTA is used as the terminating ion. Linear calibration graphs are obtained for a standard mixture of Mn(+), Cu(2+), Zn(2+), Cd(2+), Pb(2+) and Fe(3+) in the range 0.5-5.0 nmole, with relative standard deviations of 1.0% or better. The effective mobilities of the Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes increase in parallel with the stability constants, except for the Cu(II) complexes. It is concluded that the abnormal behaviour of the Cu(II) complexes may be attributed to a difference in steric configuration.

Production of catechol by transpositional mutants of aniline-assimilating Pseudomonas species AW-2

Twenty-four transpositional mutants of aniline-assimilating Pseudomonas sp. AW-2 produced catechol from aniline and accumulated it in a cultural medium. Among the 24 mutants, strain B-9 produced a maximal amount of catechol (0.97 mg/ml) from 1 mg/ml of aniline in a 13.5-h growing culture containing pyruvate and casamino acids. The conversion rate of aniline to catechol was 82% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol and citrate, and a maximal amount of catechol produced from 1 mg/ml of aniline reached 1.1 mg/ml at the conversion rate of 93% after 2 h of incubation. Enzymes catalyzing the synthesis of catechol from aniline were induced by aniline in strain B-9 cells.