Rodríguez-Salazar Julieta, Almeida-Juarez Arisbeth G, Ornelas-Ocampo Katya, Millán-López Sofía, Raga-Carbajal Enrique, Rodríguez-Mejía José Luis, Muriel-Millán Luis Felipe, Godoy-Lozano E Ernestina, Rivera-Gómez Nancy, Rudiño-Piñera Enrique, Pardo-López Liliana
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Front Microbiol. 2020 Jun 4;11:1100. doi: 10.3389/fmicb.2020.01100. eCollection 2020.
Catechol 1,2 dioxygenases (C12DOs) have been studied for its ability to cleavage the benzene ring of catechol, the main intermediate in the degradation of aromatic compounds derived from aerobic degradation of hydrocarbons. Here we report the genome sequence of the marine bacterium GOM2, isolated from the southwestern Gulf of Mexico, and the biochemical characterization of its C12DO (PsC12DO). The gene, encoding PsC12DO of 312 amino acid residues, was cloned and expressed in Many C12DOs have been described as dimeric enzymes including those present in species. The purified PsC12DO enzyme was found as an active trimer, with a molecular mass of 107 kDa. Increasing NaCl concentration in the enzyme reaction gradually reduced activity; in high salt concentrations (0.7 M NaCl) quaternary structural analysis determined that the enzyme changes to a dimeric arrangement and causes a 51% decrease in specific activity on catechol substrate. In comparison with other C12DOs, our enzyme showed a broad range of action for PsC12DO in solutions with pH values ranging from neutral to alkaline (70%). The enzyme is still active after incubation at 50°C for 30 min and in low temperatures to long term storage after 6 weeks at 4°C (61%). EDTA or Ca inhibitors cause no drastic changes on residual activity; nevertheless, the activity of the enzyme was affected by metal ions Fe, Zn and was completely inhibited by Hg. Under optimal conditions the and values were 16.13 s and 13.2 μM, respectively. To our knowledge, this is the first report describing the characterization of a marine C12DOs from isolated from the Gulf of Mexico that is active in a trimeric state. We consider that our enzyme has important features to be used in environments in presence of EDTA, metals and salinity conditions.
儿茶酚1,2-双加氧酶（C12DOs）因其能够裂解儿茶酚的苯环而受到研究，儿茶酚是烃类好氧降解产生的芳香族化合物降解过程中的主要中间体。在此，我们报告了从墨西哥湾西南部分离出的海洋细菌GOM2的基因组序列，以及其C12DO（PsC12DO）的生化特性。编码312个氨基酸残基的PsC12DO的基因被克隆并在……中表达。许多C12DOs被描述为二聚体酶，包括存在于……物种中的那些。纯化后的PsC12DO酶为活性三聚体，分子量为107 kDa。酶反应中NaCl浓度的增加会逐渐降低活性；在高盐浓度（0.7 M NaCl）下，四级结构分析确定该酶转变为二聚体排列，并导致儿茶酚底物的比活性降低51%。与其他C12DOs相比，我们的酶在pH值从中性到碱性的溶液中对PsC12DO表现出广泛的作用范围（70%）。该酶在50°C孵育30分钟后仍有活性，在4°C长期储存6周后在低温下仍有活性（61%）。EDTA或钙抑制剂对残余活性没有显著影响；然而，该酶的活性受到金属离子Fe、Zn的影响，并被Hg完全抑制。在最佳条件下，……和……值分别为16.13 s和13.2 μM。据我们所知，这是第一份描述从墨西哥湾分离出的处于三聚体状态且具有活性的海洋C12DOs特性的报告。我们认为我们的酶在存在EDTA、金属和盐度条件的环境中具有重要的应用特性。
