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首页> 美国卫生研究院文献>Journal of Bacteriology >Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite.
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Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite.

机译：细菌硝基酚氧化酶的纯化和表征该酶将邻硝基酚转化为邻苯二酚和亚硝酸盐。

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摘要

A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified. The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs. The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel). The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted. Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect. The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively. 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover. The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0. At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min. Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol.
机译：从恶臭假单胞菌B2中分离出化学计量地将邻硝基苯酚（ONP）转化为邻苯二酚和亚硝酸盐的硝基苯酚加氧酶并纯化。该酶的底物特异性很宽，包括几种卤素和烷基取代的ONP。加氧酶由一条分子量为58,000（通过凝胶过滤确定）或65,000（在十二烷基硫酸钠-聚丙烯酰胺凝胶上确定）的多肽链组成。酶促反应是NADPH依赖性的，每转化一分子ONP消耗一分子氧气。镁或锰离子刺激了酶的活性，而加入黄素腺嘌呤二核苷酸，黄素单核苷酸或还原剂则没有作用。 ONP和NADPH的表观Kms分别为8和140 microM。 2,4-二硝基苯酚竞争性地（Ki = 0.5 microM）抑制ONP转换。酶稳定性和活性的最佳pH为7.5至8.0。在40摄氏度时，酶在2分钟内完全灭活；但是，在1 mM ONP存在的情况下，即使在10分钟后仍可恢复40％的活性。在存在50％甘油的情况下，酶活性最好在-20摄氏度保存。

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