4株邻苯二甲酸二丁酯降解菌的分离鉴定及其相关降解基因的克隆

从土壤中分离纯化出4株能降解邻苯二甲酸二丁酯(DBP)的菌株,分别命名为JDC-1、JDC-8、JDC-9、JDC-12,并对其进行了形态学、生理生化及分子生物学鉴定.菌株革兰氏染色阳性,16S rDNA序列分析显示4株菌均与节杆菌属(Arthrobacter sp.)有99%以上的序列相似性,初步判断这4株菌为Arthrobacter sp..通过PCR扩增及克隆,均获得了1个约900 bp的DNA片段,测序结果显示该片段与Arthrobacter keyseri的邻苯二甲酸3,4-双加氧酶基因的核苷酸序列相似性为96%以上.对4株菌的最适生长条件及对DBP的降解能力进行了分析,结果显示,4株菌的最适生长条件为pH 7.0~8.5,温度30~35℃.以DBP作为目标测试物,在适宜条件下测试了4株菌的降解能力,显示这4株菌均为高效降解菌,效率最高的JDC-1能在28 h内将500 mg/L的DBP降解完全,最慢的JDC-8经40 h能将500 mg/L的DBP降解完全,本研究对于DBP降解机制的研究及微生物资源的开发都具有重要意义.

Isolation and Identification of Four DBP-Degrading Strains and Molecular Cloning of the Degradation Genes

Four di-butyl-phthalate(DBP)-degrading bacterial strains, JDC-1, JDC-8, JDC-9 and JDC-12, were isolated from soil. The strains were gram positive. The 16S rRNA sequence analysis revealed that the four strains had similarities of 99% with Arthrobacter sp. . According to the morphologic, physiobiochemical characteristics and the analysis of their 16S rRNA, all the four strains were identified as Arthrobacter sp. . A 900 bp DNA fragment was obtained from the four strains by PCR amplified and clone. When compared with the large subunit of phthalate dioxygenase gene (phtA) of Arthrobacter keyseri, more than 96% similarities were evident in the nucleotide sequences. The optimal growth conditions and degradation rates of DBP were tested and the result indicated that the optimal growth conditions of the four bacteria strains were pH 7.0-8.5 and 30-35℃. All the four bacteria strains performed efficiently for DBP degrading capabilities under optimal conditions. The most efficient strain JDC-1 degraded 500 mg/L DBP completely within 28 h whereas the least efficient strain JDC-8 degraded 500 mg/L DBP completely within 40 h. This study is helpful to the investigation of DBP-degrading mechanisms and the development of microbial resources.