• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.35-42
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• 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
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• 제9권3호
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• pp.249-254
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• 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.

• 생명과학회지
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• 제15권5호
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• pp.743-748
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• 2005

Histamine은 적색육 어류의 histidine이 어육 중의 Morganella morganii, Hafnia alvei 및 Klebsiella pneumoniae와 같은 부패세균에 의해 탈탄산 되어 초기에 형성되는 것으로 allergy성 식중독을 일으킬 수 있다. 이는 적색육 어류인 고등어의 선도저하 시에 많이 생성된다. 그리고 부패 후기에는 histamine을 분해하는 세균도 존재하는 것으로 알려져 있다. 그러므로, histamine 식중독의 잠재력을 지닌 자반고등어로 인한 식중독 사고 예방과 그 위생 대책을 수립하는데 필요한 자료를 얻고자 자반고등어에서 histamine 분해능을 가진 균을 분리, 동정하였다. 시료는 대형마트에서 시판되는 상태로 구입하였다. 질소원과 탄소원으로써 histamine만을 첨가한 제한배지를 사용하여 histamine 분해능을 가진 균을 분리하였다. 그리고 Cram staining, oxidase, catalase, citrate, TSI test, $H_{2}S$ reaction 및 indole 생성 등의 기본적인 생화학적 동정시험을 거쳐 10종의 시험균주를 선택하였다. 이 균주들을 16SrRNA gene 염기서열 비교에 의한 계통발생학적 분석을 이용하여 동정 하였다. 그 결과, Pseudomonas putida strain RA2, Halomonas marina, Uncultured Arctic sea ice bacterium clone ARKXV1/2-136, Halomonas venusta, Psychrobacter sp. HS5323, Pseudemonas putida KT2440, Rhodococcus erythropolis, Klebsiella terrigena (Raoultella terrigena), Alteromonadaceae bacterium T1, Shewanella massilia의 10종이 모두 동정 되 었으며, 각각 $100\%,{\;}100\%,{\;}99\%,{\;}99\%,{\;}99\%,{\;}99\%,{\;}100\%,{\;}95\%,{\;}99\%,{\;}100\%$의 상동성을 보였다. Histamine분해능의 존재를 탁도측정법과 효소법에 의해 확인한 결과, 분리된 10종 모두의 histamine 분해능이 재확인 되었고, 그 중 Shewanella massilia가 최대의 histamine 분해능을 보이는 것으로 확인되었다. 이 결과로 자반고등어 시판 제품에는 다수의 histamine 분해 세균이 존재하는 것을 확인할 수 있었으며, 이 세균을 활용한다면 식품 내 존재하는 histamine을 효과적으로 분해할 수 있을 것이라 예상된다.

• Journal of Microbiology
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• 제35권4호
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• pp.300-308
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• 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.