1,2,4-三氯苯双加氧酶和脱氢酶基因克隆与序列分析

通过PseudomonasnitroreducensJ5-1对不同氯苯类底物的降解实验,发现其降解能力大小顺序为:1,2,4-三氯苯,1,3-二氯苯,1,2-=氯苯,氯苯,与已报道的1,2,4-三氯苯降解菌株在底物利用的特性方面存在差异.采用PCR技术从J5-1中扩增获得氯苯降解过程中的关键酶--氯苯双加氧酶和脱氢酶的基因序列,分别命名为tcbA和tcbB,序列比对发现其与Burkholderia sp-PS12的氯苯双加氧酶和脱氢酶的基因序列同源性最高.通过J5-1的氯苯双加氧酶.亚基(TcbAa)与PS12的氯苯双加氧酶a亚基(TecAl)的氨基酸序列比对发现,在307-310位置有连续4个氨基酸残基的差异(1307L、M308T、1309V、Q310E),这可能是造成2株菌对1,2,4,5-四氯苯降解偏好性差异的原因.此外,通过催化芳香化合物降解的双力D氧酶a亚基的系统进化分析,认为TcbAa属于甲苯/联苯亚科,且与多取代氯苯双加氧酶e亚基的同源性最大.

Cloning and Sequence Analysis of 1,2,4-Trichlorobenzene Dioxygenase and Dehydrogenase Genes

Pseudomonas nitroreducens J5-1 is able to use monochlorobenzene, 1,2-dichlorobenzene, 1,3-dichlorobenzene and 1,2,4-trichlorobenzene as sole carbon and energy sources, and it differs from those 1,2,4-trichlorobenzene degrading bacteria reported in substrate utilizing characters. PCR technique was used to amplify the genes of chlorobenzene dioxygenase and dehydrogenase of J5-1, and they were named as tcbA and tcbB, respectively. Homology analysis indicated that these genes and gene products were most closely related to those of Burkholderia sp. PS12. By alignment of the amino acid sequences of the a subunits of TcbAa (from J5-1) and TecA1 (from PS12), four amino acid residues from site 307 to site 310 were found to be different (I307L, M308T, I309V, Q310E), which probably retarded the preference for the substrate 1,2,4,5-tetrachlorobenzene. Furthermore, the phylogenetic analysis of the dioxygenase alpha subunits showed that TcbAa was belong to the toluene/diphenyl subfamily, and was most closely related to the poly-chlorinated benzene dioxygenase alpha subunit.