Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of α-class in cyanobacterial cells. The enzyme activity was confirmed both in vivo in intact cells and in vitro, using the recombinant protein. CA activity in intact cells of Cyanothece sp. ATCC 51142 reached ∼0.6 Wilbur–Anderson units (WAU) per 1 mg of total cell protein, and it was inhibited by a specific CAs inhibitor, ethoxyzolamide. The genes cce_4328 (ecaA) and cce_0871 (ecaB), encoding two potential extracellular CAs of Cyanothece have been cloned, and the corresponding proteins EcaA and EcaB, representing CAs of α- and β-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (∼1.1 × 10⁴ WAU per 1 mg of target protein) was detected for the recombinant EcaA only. The presence of EcaA in the outer cellular layers of Cyanothece was confirmed by immunological analysis with antibodies raised against the recombinant protein. The absence of redox regulation of EcaA activity indicates that this protein does not possess a disulfide bond essential for some α-class CAs. The content and activity of EcaA in a fraction of periplasmic proteins was higher in Cyanothece cells grown at ambient concentration of CO₂ (0.04%) compared to those grown at an elevated CO₂ concentration (1.7%). At the same time, the level of ecaA gene mRNA varied insignificantly in response to changes in CO₂ supply. Our results indicate that EcaA is responsible for CA activity of intact Cyanothece cells and point to its possible physiological role under low-CO₂ conditions.

在这里，我们首次报道了在蓝细菌细胞中存在α类高活性细胞外碳酸酐酶（CA）。使用重组蛋白在体内和体外均证实了酶活性。Cyanothece sp。完整细胞中的CA活性。每1 mg总细胞蛋白，ATCC 51142达到〜0.6 Wilbur-Anderson单位（WAU），并且被一种特定的CAs抑制剂乙氧基唑酰胺抑制。基因cce_4328（ECAA）和cce_0871（ECAB），编码的两个潜在的细胞外的CA蓝丝已克隆了相应的蛋白质，分别代表α-和β-类CA的相应蛋白质EcaA和EcaB已在大肠杆菌中异源表达。仅对重组EcaA检测到高的比活（每1 mg靶蛋白约1.1×10 ⁴ WAU）。通过针对重组蛋白的抗体的免疫学分析证实了蓝藻细胞外层中存在EcaA。缺乏对EcaA活性的氧化还原调节表明，该蛋白不具有某些α类CA必需的二硫键。在周围CO ₂浓度下生长的蓝藻细胞中，一部分周质蛋白中EcaA的含量和活性较高。（0.04％）与在较高的CO ₂浓度下生长的那些（1.7％）相比。同时，ecaA基因mRNA的水平随CO ₂供应的变化而变化不大。我们的结果表明，EcaA负责完整蓝藻细胞的CA活性，并指出在低CO ₂条件下其可能的生理作用。