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研究报告

Isolation and characterization of norfloxacin-degrading bacterium strain NOR-36

摘要：利用微生物分离筛选技术从医药厂废水池活性污泥中分离得到一株可以以哌嗪为一级基质共代谢降解诺氟沙星的菌株.经生理生化鉴定和16S rDNA基因序列同源性分析，将此菌株初步鉴定为山羊葡萄球菌（Staphylococcus caprae），命名为NOR-36.当菌株NOR-36在温度为30℃、pH 7.0、诺氟沙星起始浓度为5 mg·L^-1，哌嗪与诺氟沙星浓度比为5：1时培养10 d降解率可达92.6%.而在相同的实验条件下，不加一级基质的空白实验中诺氟沙星的降解率最高仅为8.5%.可知菌株NOR-36以哌嗪为一级基质对诺氟沙星进行共代谢生物降解，其对诺氟沙星的降解率显著提高.采用高效液相色谱-质谱联用仪分析确定菌株降解诺氟沙星的过程中产生3种主要的中间代谢产物，分子式分别为：C₁₆H₁₈FN₃O₄（产物1）、C₁₄H₁₆FN₃O₃（产物2）、C₁₂H₁₁FN₂O₃（产物3）.分析中间产物结构可知：NOR-36首先在共代谢关键酶作用下使诺氟沙星哌嗪环上碳氮键断裂，并且中间位置碳原子被氧原子氧化，加氧形成羰基，得到产物1；之后产物1继续被氧化，碳氮键继续断裂，-COCH₃基团被去除生成产物2，最后产物2经氧化后，-CH₂CH₂NH₂基团被去除，最终得到产物3.

Abstract：In the present study, a strain was isolated from pharmaceutical wastewater treatment pool to degrade norfloxacin by using piperazine as a co-metabolic substrate. According to the phenotype, physiological and biochemical analysis, the strain was preliminary identified as Staphylococcus caprae and designated as NOR-36 The maximum degradation efficiency (92.6%) of norfloxacin with an initial concentration of 5 mg·L^-1 by NOR-36 could be achieved after 10 d under the conditions as follows:temperature 30℃, pH 7.0, concentration of piperazine 25 mg·L^-1 (piperazine:norfloxacin=5:1). When comparing with the blank test (no piperazine), the results clearly indicate that piperazine could be served as a co-metabolic substrate for the degradation of norfloxacin by NOR-36. Three intermediates were structurally, C₁₆H₁₈FN₃O₄ (product 1), C₁₄H₁₆FN₃O₃ (product 2) and C₁₂H₁₁FN₂O₃ (product 3), respectively, which were identified by using the HPLC-MS/MS analysis. Based on the structures of the intermediates, the probable degradation pathway of norfloxacin by NOR-36 was tentatively proposed. The C-N on the piperazine ring of norfloxacin was firstly broken and oxidized. then, -COCH₃ and -CH₂CH₂NH₂ group were removed subsequently.

Key words：Staphylococcus caprae NOR-36 norfloxacin co-metabolism level of substrate metabolite