Abstract: The composition and expression pattern of the car gene cluster were studied using carbazole-degrading bacterial Sphingosinicella sp. strain B1 as material, and the key enzyme mechanism was explained in carbazole degradation process. By analyzing the conserved regions of the genes in the car gene cluster of different strains, carAa, carBa, carBb and carC genes were successfully cloned from bacterial strain B1. The RT-PCR test confirmed that the car gene cluster of bacterial strain B1 was induced expression. The degraded key gene carB, and 2 calveolin genes carBa and carBb were homologously cloned and heterologously expressed in E.coli. The enzymatic properties of CarB were analyzed by catalyzing 2,3-dihydroxybiphenyl. The results showed that the optimal pH value and temperature of CarB were 7.4 and 30℃, respectively, and Fe2+ could significantly improve the enzyme catalytic activity.

Key words: carbazole, Sphingosinicella sp., degradation gene, enzymatic properties

摘要： 以分离到的咔唑高效降解菌鞘氨醇胞菌B1为材料，研究该菌株咔唑降解基因簇的组成和表达模式，阐释咔唑降解过程中关键酶的作用机理。通过分析不同菌株咔唑降解基因簇中基因的保守区，从菌株B1中成功克隆得到基因簇中carAa、carBa、carBb和carC基因片段。通过RT-PCR证实菌株B1的咔唑降解基因簇为诱导表达。通过同源克隆得到降解关键基因carB和编码两个亚基的carBa、carBb基因，并在大肠杆菌中进行了异源表达。以2,3-二羟基联苯为底物，对CarB粗酶液进行了酶学性质分析。研究表明CarB蛋白催化最适pH和温度分别为7.4和30℃，Fe2+能显著提高酶的催化活力。

关键词: 咔唑, 鞘氨醇胞菌, 降解基因, 酶学性质