Characterization of Catechol l,2-Dioxygenase Purified from the Benzoate Degrading Bacterium, Pseudomonas sp. NFQ-l Isolated from Dead Coal Pit Areas

Our previous research has demonstrated that the bacterium, Pseudomonas sp. NFQ-l capable of utilizing quin­oline (2,3-benzopyridine) as the sole source of carbon, nitrogen, and energy was isolated and characterized [Yoon et ai. (2003) Kor. J. Biotechnol. Bioeng. 18(3):174-179]. In this study, we have found that Pseudomonas sp. NFQ-l could degrade quinoline as well as benzoate, and extended this work to characterize the catechol 1,2­dioxygenase (C1,2O) purified from the bacterium cultured in benzoate media. Initially, C1,2O has been purified by ammonium sulfate precipitation, gel permeation chromatography, and Source 15Q. After Source 15Q, puri­fication fold was increased to approximately 14.21 unit/mg. Molecular weight of C1,2O was about 33 kDa. Physicochemical characteristics (e.g., substrate specificity, Km, Vmax, pH, temperature and effect of inhibitors) of purified C1,2O were examined. C1,2O demonstrated the activity for catechol, 4-methylcatechol and 3-meth­ylcatechol as a substrate, respectively. The Km and Vmax value of C1,2O for catechol was 38.54 ${\mu}M$ and $25.10\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}.$ The optimal temperature of C1,2O was $30^{\circ}C$ and the optimal pH was approximately 8.5. Metal ions such as $Ag^+,\;Hg^+,\;Ca^{2+},\;and\;Cu^{2+}$ show the inhibitory effect on the activity of C1,2O. N-terminal amino sequence of C1,2O was analyzed as ^1TVKISQSASIQKFFEEA^{17}.$ In this work, we found that the amino acid sequence of NFQ-l showed the sequence homology of 82, 71, 59 and $53\%$ compared with C1,2O from Pseudomonas aeruginosa PA0l, Pseudomonas arvilla C-1., P. putida KT2440 and Pseudomonas sp. CA10, respectively.

폐광지역에서 분리한 Benzoate 분해세균 Pseudomonas sp. NEQ-1에서 정제된 Catechol 1,2-Dioxygenase의 특성

Quinoline (2,3-benzopyridine)을 유일한 탄소원과 질소원, 그리고 에너지원으로 이용하는Pseudomonas sp. NEQ-1을 실험 균주로 사용하였으며, 균주로부터 catechol 1,2-dioxygenase (C1,2O)를 유도하기 위하여 탄소원으로 benzoate를사 용하였다. C1,2O의 효소학적 특징을 조사하기 위하여 benzoate에서 배양한 Pseudomonas sp. NFQ-1을 초음파 분쇄기로 파쇄하고, ammonium sulfate침전과 gel permeation chromatography및 Source 15Q의 과정을 실시하여 C1,2O를 분리 및 정제하였다. 정제된 C1,2O의 특이활성(specific activity)은 14.21 unit/mg으로 나타났으며, SDS-PAGE에 의해 조사된 C1,2O의 분자량은 약 33 kDa이었다. Cl,2O는 catechol과 4-methylcatechol 및 3-methylcatechol에 대해서 효소활성을 나타내는 것으로 확인되었다. C1,2O의 Km은 38.54 ${\mu}M$로 측정되었고, Vmax는 $25.10\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}$으로 나타났다. C1,2O는 $30^{\circ}C$와 pH 8.5에서 최적활성을 나타내는 것으로 조사되었으며, $Ag^+,\;Hg^+,\;Ca^{2+}$,그리고 $Cu^{2+}$는 C1,2O의 활성을 억제하였다. 분석되어진 N-말단 아미노산 서열은 ^1TVKISQSASIQKFFEEA^{17}$이었으며, Pseudomonas aeruginosa PA01과 $82\%$로 가장 높은 유사성을 보였고 Pseudomonas arvilla C-1와는 $71\%,$ Pseudomonas putida KT2440과는 $59\%,$ 그리고 Pseudomonas sp. CA10과는 $53\%$의 상동성이 각각 존재하는 것으로 확인하였다.