菲降解菌的筛选鉴定及其降解酶基因的研究

ISOLATION OF PHENANTHRENE-DEGRADING BACTERIA ANDANALYSIS OF THEIR DEGRADING-ENZYME GENE

经过富集从活性污泥中筛选出两株菲降解菌WSCII和WSCIII,经16SrDNA鉴定,它们分别属于鞘氨醇单胞菌属(Sphingomonassp. )和假单胞菌属(Pseudomonassp. ),在28℃下4d内对菲( 0. 6g/L)的降解率分别达到了70. 9%和78. 1%;在LB、萘和菲为碳源的培养条件下,测得菌体的双加氧酶比活力不同.在菲的诱导下,两株菌的双加氧酶比活力最高,而以LB和萘为碳源时酶的比活力较低,其中WSCII在萘中不能生长;这表明该酶在两菌中皆为底物诱导表达.以其它菌的萘双加氧酶基因大亚基片段做模板制备异源探针,与两菌总DNA进行斑点杂交;结果表明,这两株菌可能含有不同的菲双加氧酶基因.利用简并引物进行PCR扩增菲双加氧酶,结果仅能从WSCIII的总DNA中扩增得到目的片段,序列分析证明,该基因与已报道的(P. putidaNCIB9816 -4, AF491307 )双加氧酶同源性最高为98%.

Two phenanthrene-degrading bacteria, WSCII and WSCIII, were isolated from the activated sludge of a city sewage treatment factory. Based on 16S rDNA sequence, they were supposed belonging to Sphingomonas sp. and Pseudomonas sp. Under 28 ℃, the two strains degraded phenanthrene (0.6 g/L) by 70.9% and 78.1% within 4 days,respectively. Different dioxygenase activities were detected in both strains when cultivated on different carbon sources, such as LB, naphthalene and phenanthrene. The highest was in the case of phenanthrene, which indicated that the expression of dioxygenase could be induced by its specific substrate. Dot blotting showed positive hybridization of both strains' total DNA with a dioxygenase heterogeneous dioxygenase probe, but might have different similarities indicated by different signal strength. A fragment of dioxygenaseα-subunit gene was obtained by a degenerate PCR from the total DNA of WSCIII, it shared 98% similarity with the dioxygenase of P. putida NCIB9816-4(AF491307). Fig 5, Ref 10