• 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.9 no.3
• /
• pp.249-254
• /
• 1999

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.

• Journal of Life Science
• /
• v.15 no.5 s.72
• /
• pp.743-748
• /
• 2005

Histamine can be produced at early spoilage stage through decarboxylation of histidine in red-flesh fish by Proteus morganii, Hafnia alvei or Klebsiella pneumoniae. Allergic food poisoning is resulted from the histamine produced when the freshness of Mackerel degrades. Conversely it has been reported that there are bacteria which decompose histamine at the later stage. We isolated histamine decomposers from salted mackerel and studied the characteristics to help establish hygienic measure to prevent outbreak of salted mackerel food poisoning. All the samples were purchased through local supermarket. Histamine decomposers were isolated using restriction medium using histamine 10 species were selected. Identification of these isolates were carried out by the comparison of 16S rDNA partial sequence; as a result, we identified Pseudomonas putida strain RA2 and Halomonas marina, Uncultured Arctic sea ice bacterium clone ARKXV1/2-136, Halomonas venusta, Psychrobacter sp. HS5323, Pseudomonas putida KT2440, Rhodococcus erythropolis, Klebsiella terrigena (Raoultella terrigena), Alteromonadaceae bacterium T1, Shewanella massilia with homology of $100\%,{\;}100\%,{\;}99\%,{\;}99\%,{\;}99\%,{\;}99\%,{\;}100\%,{\;}95\%,{\;}99\%,{\;}and{\;}100\%$respectively. Turbidometry determination method and enzymic method were employed to determine the ability of histamine decomposition. Among those species Shewanella massilia showed the highest in ability of histamine decomposition. From these results we confirmed various histamine decomposer were present in salted mackerel product in the market.

• Journal of Microbiology
• /
• v.35 no.4
• /
• pp.300-308
• /
• 1997

The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.