【作者】 吕鹏；

【导师】 张长铠；

【作者基本信息】 山东大学 ， 微生物学， 2006， 博士

【摘要】 随着经济的迅猛发展，引发的环境问题倍受关注。目前我国环境污染严峻，特别是芳香族污染物在土壤和水中的含量急剧上升，严重危及到了人们的生存。微生物的降解是芳香族污染物脱毒的最有效手段，亦是最终途径。因此，从环境中筛选高效的芳香族污染物降解菌，研究其降解途径关键酶及其基因的性质与特点，并将其应用到环境问题中去，具有重要的理论意义与实际意义。 本文从环境水体中分离到了多株苯酚降解菌，经过复筛，选择能够高效降解苯酚的L68菌株进行研究。经形态及生理生化鉴定与16S rDNA序列分析，从多相分类的角度并融合细菌系统分类最新研究成果，将L68菌株鉴定为Burkholderia cepacia（洋葱伯克霍尔德氏菌、洋葱伯克氏菌、洋葱球茎病伯克氏菌）。对洋葱伯克霍尔德氏菌L68菌株的苯酚降解性能进行了测定，研究结果显示，该菌株适宜生长温度为35℃左右。适宜生长pH值为7.0左右。在苯酚浓度为0.5g/L的苯酚无机盐液体培养基中35℃、160rpm摇瓶振荡培养L68菌株24h后，用4-氨基安替比啉法不能检测到酚。当培养基中初始苯酚浓度为0.8g/L时，L68菌株还能够保持一定的对苯酚的降解能力。L68菌株对苯酚的降解作用主要发生在指数生长期，菌体的生长与苯酚的降解之间呈现正相关。固定化可以提高L68菌株对苯酚的抗性。当培养基中初始苯酚浓度高达1.5g/L时，固定化L68菌株仍然可以对苯酚进行降解。 实验证实，洋葱伯克霍尔德氏菌L68菌株只产生邻苯二酚2，3-双加氧酶，通过间位裂解途径使邻苯二酚及其衍生物开环。本文首次报道了来源于伯克霍尔德氏菌的邻苯二酚2，3-双加氧酶的纯化与一些酶活性质。通过正交实验优化了该菌株的产酶培养条件，适宜产酶培养条件确定为1‰的苯酚含量，500ml三角瓶中250mi的装液量，0.2‰的酵母粉含量，培养基初始pH值6.0，接种量2.5％。L68菌株细胞抽提液经硫酸铵分级沉淀、Sephadex G-150柱层析和DEAE-Sepharose Fast Flow柱层析后，经SDS-PAGE检测，达到了电泳纯，提纯倍数为309.66倍，收率为55.43％。测得该菌株所产邻苯二酚2，3-双加氧酶亚基分子量为34±1kDa，等电点为4.2。邻苯二酚和4-甲基邻苯二酚的Km值分别为4.9μM和9.9μM，是酶的良好底物。L68菌株邻苯二酚2，3-双加氧酶在紫外光谱吸收上具有普通蛋白的共性。荧光光谱分析表明，酶显示出很强的荧光，最大发射波长λ_emmax为更多还原

【Abstract】 Modern industry produced a variety of aromatic compounds and resulted in the environmental pollution. Bacterial expressing the enzymes of the pathways for the degradation of aromatic compounds were used to cleaning up contaminated environments.A strain that can grow on phenol as the sole source of carbon and energy, L68, was isolated, and identified as Burkholderia cepacia. The phenol-degradation conditions of the bacteria were analyzed. The optimal growth temperature is 35°C and the optimal growth pH value is 7.0. No phenol was detected after the strain growing on the inorganic salt medium containing 0.5g/L of phenol at 35°C for 24h. Strain L68 was capable of degrading catechol when the phenol concentration reached 0.8g/L. Immobilized strain L68 could degrade phenol when the phenol concentration was as high as 1.5g/L.Strain L68 was capable of degrading catechol via the meta-cleavage pathway. In this thesis, the purification and characteristics of catechol 2,3-dioxygenase from Burkholderia were reported for the first time. Culture conditions improvement for the production of catechol 2,3-dioxygenase by strain L68 was investigated using orthogonal array designs. The experimental results revealed that the volume of medium and the percent of phenol had remarkable effects on the enzyme production, while the percent of inoculation played little role in the production of the enzyme. The improved culture conditions are 250 ml medium consisted of 0.2‰ yeast extract and 19‰ phenol, pH 6.0, and 2.5 % inoculation. Catechol 2,3-dioxygenase was purified to homogeneity from the cell extract of the strain by ammonium sulfate-fraction, Sephadex G-150 and DEAE-Sepharose Fast Flow column chromatography. The specific activity of the purified enzyme was 309.66 folds as high as that of crude extract and the yield was about 55.43%. The molecular mass of enzyme subunit is 34±lkDa and the pI was about 4.2. When catechol and 4-methyl-catechol were used as substrate, the Km values of the enzyme were 4.9μM and 9.9μM, respectively. The UV spectrum of catechol 2,3-dioxygenase from L68 was the same as most of the protein and the fluorescence spectrum showed the λ _emmax was 336nm. At 20°C, the circular dichroism spectrum showed that the contents of α-helix, β-sheet, β-turn and random coil were 45.8%, 15.6%, 23.4% and 15.3%, respectively. The dependence of更多还原