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为了筛选得到一株产胞外邻苯二酚-2,3-双加氧酶的芘降解菌株,以新疆克拉玛依石油污染土壤为样品源,采用芘平板升华法,筛选具有多环芳烃降解能力的菌株。利用显色反应及酶促反应对菌株所产胞外邻苯二酚-2,3-双加氧酶进行定性定量试验并对其进行形态学观察、BIOLOG GENⅢ微孔板鉴定及16S rRNA序列分析。通过单因子影响试验和正交试验对菌株的生长特性及最佳降解条件进行了初步探讨。结果表明:芘降解菌株W39能分泌胞外邻苯二酚-2,3-双加氧酶,且经芘诱导后,产酶能力提高了3倍;经鉴定,菌株W39为阴沟肠杆菌(Enterobacter cloacae);最适培养条件:35℃,p H 7.0,芘浓度50 mg/L。可见,邻苯二酚-2,3-双加氧酶是降解芘的关键酶之一,可对其进行进一步研究。
In order to obtain a pyrene-degrading strain with extracellular catechol-2,3-dioxygenase, the petroleum contaminated soil of Karamay in Xinjiang was used as the sample source. Pyrene plate sublimation method was used to screen the pyrene-degrading strain with polycyclic aromatic hydrocarbons Strain. The colorimetric reaction and enzymatic reaction were used for qualitative and quantitative analysis of the extracellular catechol-2,3-dioxygenase produced by the strain. Morphological observation, BIOLOG GENⅢ microplate identification and 16S rRNA sequence analysis . The growth characteristics of the strain and the optimum degradation conditions were studied by single-factor impact test and orthogonal test. The results showed that the pyrene-degrading strain W39 secreted extracellular catechol-2,3-dioxygenase and its ability to produce enzymes increased by 3-fold after induced by pyrene. The strain W39 was identified as Enterobacter cloacae). The optimum culture conditions were 35 ℃, p H 7.0 and pyrene concentration 50 mg / L. Visible, catechol -2,3-dioxygenase pyrene degradation of one of the key enzymes, which can be further studied.