鞘氨醇胞菌B1咔唑降解基因carB的克隆与功能研究

Cloning and Functional Studies of Carbazole Degradation Gene carB from Sphingosinicella sp. Strain B1

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car Aa、car Ba、car Bb和car C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car B和编码两个亚基的car Ba、car Bb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Car B粗酶液进行了酶学性质分析。研究表明Car B蛋白催化最适p H和温度分别为7.4和30℃,Fe2+能显著提高酶的催化活力。

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,car Aa,car Ba,car Bb and car C 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 car B,and 2 calveolin genes car Ba and car Bb were homologously cloned and heterologously expressed in E.coli. The enzymatic properties of Car B were analyzed by catalyzing 2,3-dihydroxybiphenyl. The results showed that the optimal p H value and temperature of Car B were 7. 4 and 30℃,respectively,and Fe2＋could significantly improve the enzyme catalytic activity.