芳香烃化合物的微生物降解及基因工程菌的构建
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摘要
本研究以苯酚为唯一的碳源和能源从农药厂活性污泥和煤气厂污水排放口周围的土壤中分离到三株降解能力较好的革兰氏阴性的芳香烃降解菌，分别命名为ZD 2、ZD 4-1和ZD 4-3。通过细菌的16S rDNA序列比对分析以及相关形态学和生理生化特性分析，将ZD 2、ZD4-1和ZD4-3三株菌分别鉴定属于多食鞘氨醇杆菌(Sphingobacterium multivorum)，睾丸酮丛毛单胞菌(Comamonas testosteroni)和铜绿假单胞菌(Pseudomonas aeruginosa)。基于16S rDNA序列的系统分类研究表明，菌株ZD4-1和ZD4-3分别属于两个不同的分类亚组。
研究了Sphingobacterium multivorum ZD 2对五氯酚(PCP)的共代谢降解。结果表明，苯酚作为生长底物对细菌的生长有一定的抑制作用，PCP共代谢降解效率低。以葡萄糖为生长底物时PCP降解效果更好，当葡萄糖的量能够满足细菌的生长以及提供降解PCP所需的还原性物质的量时，则生长底物不再是PCP降解的限制性因素，继续增加葡萄糖不能促进PCP的降解，葡萄糖和PCP之间不存在底物的竞争性抑制现象。蛋白质电泳结果表明，葡萄糖为生长底物时，PCP的降解酶是由非生长底物PCP本身所诱导，它们不同于细菌利用葡萄糖的酶。GC-MS分析PCP共代谢降解中间产物结果表明，在好氧降解情况下，PCP的共代谢降解可能也存在渐次脱氯生成苯酚并最终被矿化的过程。
以菌株Comamonas testosteroni ZD4-1和Pseudomonas aeruginosa ZD4-3为材料，研究了它们对芳香烃化合物的降解机理。结果表明，ZD4-1和ZD4-3分别利用邻苯二酚1,2-双加氧酶催化的邻裂途径和邻苯二酚2,3-双加氧酶催化的间裂途径降解苯酚。邻苯二酚1,2和2,3-双加氧酶都是可诱导的双加氧酶，其活性强烈的依赖于其降解底物的出现。细菌ZD4-3中的间裂途径对苯酚的降解效率要高于菌株ZD4-1的邻裂途径，但是后者降解苯酚的适宜pH值范围以及对芳香烃化合物利用的基质谱范围宽于前者。
通过质粒消除、PCR扩增以及Southern杂交等方法将Pseudomonas aerugionas ZD4-3中的邻苯二酚2,3-双加氧酶基因(pheB)定位于细菌染色体上。采用PCR扩增的方法从ZD4-3菌中克隆得到大小为0.9kb左右的pheB基因。测序结果表明，pheB基
因全长924bp，编码由307个氨基酸组成的邻苯二酚2，3一双加氧酶。利用表达载体pET22b
(+)对pheB进行了表达，酶活性检测结果表明，大肠杆菌粗酶提取液中的酶活性为
3.5617U/mg蛋白质，明显高于野生菌ZD4一3中的酶活性。
为了解pheB基因编码的邻苯二酚2，3一双加氧酶序列与功能之间的关系，利用生物
信息学软件对其结构与功能进行了分析。在GenBank中搜索得到相关的11个外二元醇
双加氧酶的序列，用 Bioedit软件进行同源比对分析发现，在全序列中共有12个氨基酸
同源区域，它们主要分布在氨基酸的C一末端和N一末端，而且甘氨酸同源的几率出现较
多。根据对已知的外二元醇双加氧酶的晶体结构分析推断，外二元醇双加氧酶的Gly一31、
His一157、Leu一184很可能是形成酶的催化活性中心结构域，而C一末端和N一末端的保守
序列可能是酶的结构域形成中心。催化单环芳烃裂解的双加氧酶和催化双环芳烃裂解的
双加氧酶之间的同源性比值较低，遗传距离上较远，它们之间存在明显的差别，而常温
性的C23O与耐热的C23O在氨基酸同源性方面没有显示出明显的差别。
为了方便基因工程菌监测，将邻苯二酚2，3一双加氧酶基因pheB和绿色荧光蛋白编
码基因肺融合后同时导入到受体菌中。采用一种不需要限制核酸酶和连接酶的新方法
—“三引物PcR一步法”(TP一PCR)将pheB基因和肺基因融合，构建得到户g融
合蛋白基因。测序结果表明，户g基因的5’端为pheB基因序列，3’端为加基因序列，
中间为柔性肤段一Gly4Ser-的序列，符合预期结果。为验证融合蛋白基因的功能，对户g
基因在E.colt中进行了表达实验。结果表明，蛋白质电泳出现大小约64KD的蛋白条带、
荧光显微镜检测到细菌发出的绿色荧光，IPTG诱导后检测到破碎上清液中邻苯二酚2，3-
双加氧酶酶活性为3.2224U/mg蛋白。以上结果说明，户g基因得到了表达，表达的融
合蛋白具有绿色荧光蛋白和邻苯二酚2，3一双加氧酶的活性。这为将户g基因导入野生菌
中构建到芳香烃降解基因工程菌奠定了基础。
将乡g基因重组到叫Tmini一Tns转座子载体pU下Hg中，构建了一个户g基因的转
基因载体pUT-Hg:fPg。利用双亲接合转移技术将户g基因插入到受体菌c口ma用onas
testosteroni ZD4一1的染色体中，并筛选到一株转基因工程菌Comamonas testostero爪
ZD4一1:加g。ZD4一卜加g在荧光显微镜下受480nxn紫外光激发能够发出绿色荧光，但是
办瞥基因中pheB基因表达的邻苯二酚2，3一双加氧酶活性比较弱，不很稳定，有待后续
工作中进一步完善。基因稳定性试验表明，户g基因能够在ZD4一1-fPg中稳定的存在，
由于ZD4一卜加g在480nm紫外光激发下能发出绿色荧光，因此该菌能够作为生物修复
的示踪菌，在污染环境生物修复中具有潜在的应用价值。
Three aromatic compounds degrading bacterial strains ZD 2, ZD 4-1 and ZD 4-3 were isolated from the sludge of pesticide manufacturing factory and the soil around the coal gas factory. All these isolates are Gram-negative, aerobic, non-spore-forming rods. Based on phenotypic characteristics and 16S rDNA sequence alignment, strains ZD 2, ZD 4-1 and ZD 4-3 were identified as Sphingobacterium multivorum, Comamonas testosteroni and Pseudomonas aeruginosa, respectively. The result of phylogenetic analysis showed that strain ZD 4-1 and ZD4-3 were positioned in two different subclusters. Cometabolism of pentachlorophenol (PCP) was studied by using Sphingobacterium multivorum ZD 2 to investigate the metabolism mechanism of PCP. The effect of PCP degradation with the phenol or glucose as growth substrates was compared. The results revealed that not only the PCP but also the phenol inhibited the bacterial cells and glucose was better than the phenol as a growth substrate for PCP cometabolism because there was no compet
itive inhibition between the glucose and PCP. Protein electrophoresis results showed that the unspecific enzymes for PCP degradation were induced by the non-growth substrate PCP itself when glucose was served as growth substrate. Gas chromatograph-Mass (GC-MS) analysis indicated that phenol was one of intermediate products during PCP cometabolism, suggesting that the dechlorination reaction increasingly happened even on the condition of aerobic biodegradation process.
To investigate the characteristic and biochemical mechanism about the biodegradation of aromatic compounds by bacteria, bacterial strains ZD 4-1 and ZD 4-3 were used to degrade phenol. The identification of the intermediates and the detection of the corresponding catabolic enzymes in crude extracts indicated that the strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and wefar-pathways, respectively. The results of induction experiment showed that the catechol dioxygenases, both catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O), were all inducible. Finally, the results of biodegradation and enzyme assays showed that the biodegradation efficiency of phenol by meta-Tpathways was higher than that by ortho-pathways but the former is in an inferiority
position in terms of adaptation to pH fluctuation and of growth ability on other aromatic compounds compared with the latter.
C23O is one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta-position of dihydroxylated aromatic substrates. C23O is able to catalyze the conversion of catechol to 2-hydroxymuconic semialdehyde (HMS). Based on curing experiment, PCR identification and Southern Hybridization, the C23O encoding gene pheB was localized on a 3.5 kb EcoRI/BamHI fragment and was then cloned from P. aeruginosa ZD 4-3 that was able to degrade both single and bicyclic compounds via a meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it has one open reading frame (ORF) encoding 307 amino acids, which showed a strong overall amino acid similarity to the known Gram-negative bacterial mesophilic C23Os. The heterogonous expression of the pheB gene in E. coll BL21 indicated that the activity of C23O was 3.5617U/mg protein and was higher that that of the C23O in wild bacterium.
In order to probe the relationship of the structure and function of C23O encoded by pheB gene, the alignment was performed by comparing the homology between the pheB deduced amino acids sequence and the other extradiol dioxygenases by the Bioinformatics software. The alignment analysis showed that 12 homology areas aroused during the whole sequences and these homology areas mainly distributed near the C-end and N-end. According to three-dimension crystal structure of the known extradiol dioxygenases, the amino acids Gly-31, His-157, Leu-184 were speculated to be the active sites of the C23O in this study. Moreover, there are evident differences between the extradiol dioxygenases which degrade single ring aromatic compounds, and those bicyclic
研究了Sphingobacterium multivorum ZD 2对五氯酚(PCP)的共代谢降解。结果表明，苯酚作为生长底物对细菌的生长有一定的抑制作用，PCP共代谢降解效率低。以葡萄糖为生长底物时PCP降解效果更好，当葡萄糖的量能够满足细菌的生长以及提供降解PCP所需的还原性物质的量时，则生长底物不再是PCP降解的限制性因素，继续增加葡萄糖不能促进PCP的降解，葡萄糖和PCP之间不存在底物的竞争性抑制现象。蛋白质电泳结果表明，葡萄糖为生长底物时，PCP的降解酶是由非生长底物PCP本身所诱导，它们不同于细菌利用葡萄糖的酶。GC-MS分析PCP共代谢降解中间产物结果表明，在好氧降解情况下，PCP的共代谢降解可能也存在渐次脱氯生成苯酚并最终被矿化的过程。
以菌株Comamonas testosteroni ZD4-1和Pseudomonas aeruginosa ZD4-3为材料，研究了它们对芳香烃化合物的降解机理。结果表明，ZD4-1和ZD4-3分别利用邻苯二酚1,2-双加氧酶催化的邻裂途径和邻苯二酚2,3-双加氧酶催化的间裂途径降解苯酚。邻苯二酚1,2和2,3-双加氧酶都是可诱导的双加氧酶，其活性强烈的依赖于其降解底物的出现。细菌ZD4-3中的间裂途径对苯酚的降解效率要高于菌株ZD4-1的邻裂途径，但是后者降解苯酚的适宜pH值范围以及对芳香烃化合物利用的基质谱范围宽于前者。
通过质粒消除、PCR扩增以及Southern杂交等方法将Pseudomonas aerugionas ZD4-3中的邻苯二酚2,3-双加氧酶基因(pheB)定位于细菌染色体上。采用PCR扩增的方法从ZD4-3菌中克隆得到大小为0.9kb左右的pheB基因。测序结果表明，pheB基
因全长924bp，编码由307个氨基酸组成的邻苯二酚2，3一双加氧酶。利用表达载体pET22b
(+)对pheB进行了表达，酶活性检测结果表明，大肠杆菌粗酶提取液中的酶活性为
3.5617U/mg蛋白质，明显高于野生菌ZD4一3中的酶活性。
为了解pheB基因编码的邻苯二酚2，3一双加氧酶序列与功能之间的关系，利用生物
信息学软件对其结构与功能进行了分析。在GenBank中搜索得到相关的11个外二元醇
双加氧酶的序列，用 Bioedit软件进行同源比对分析发现，在全序列中共有12个氨基酸
同源区域，它们主要分布在氨基酸的C一末端和N一末端，而且甘氨酸同源的几率出现较
多。根据对已知的外二元醇双加氧酶的晶体结构分析推断，外二元醇双加氧酶的Gly一31、
His一157、Leu一184很可能是形成酶的催化活性中心结构域，而C一末端和N一末端的保守
序列可能是酶的结构域形成中心。催化单环芳烃裂解的双加氧酶和催化双环芳烃裂解的
双加氧酶之间的同源性比值较低，遗传距离上较远，它们之间存在明显的差别，而常温
性的C23O与耐热的C23O在氨基酸同源性方面没有显示出明显的差别。
为了方便基因工程菌监测，将邻苯二酚2，3一双加氧酶基因pheB和绿色荧光蛋白编
码基因肺融合后同时导入到受体菌中。采用一种不需要限制核酸酶和连接酶的新方法
—“三引物PcR一步法”(TP一PCR)将pheB基因和肺基因融合，构建得到户g融
合蛋白基因。测序结果表明，户g基因的5’端为pheB基因序列，3’端为加基因序列，
中间为柔性肤段一Gly4Ser-的序列，符合预期结果。为验证融合蛋白基因的功能，对户g
基因在E.colt中进行了表达实验。结果表明，蛋白质电泳出现大小约64KD的蛋白条带、
荧光显微镜检测到细菌发出的绿色荧光，IPTG诱导后检测到破碎上清液中邻苯二酚2，3-
双加氧酶酶活性为3.2224U/mg蛋白。以上结果说明，户g基因得到了表达，表达的融
合蛋白具有绿色荧光蛋白和邻苯二酚2，3一双加氧酶的活性。这为将户g基因导入野生菌
中构建到芳香烃降解基因工程菌奠定了基础。
将乡g基因重组到叫Tmini一Tns转座子载体pU下Hg中，构建了一个户g基因的转
基因载体pUT-Hg:fPg。利用双亲接合转移技术将户g基因插入到受体菌c口ma用onas
testosteroni ZD4一1的染色体中，并筛选到一株转基因工程菌Comamonas testostero爪
ZD4一1:加g。ZD4一卜加g在荧光显微镜下受480nxn紫外光激发能够发出绿色荧光，但是
办瞥基因中pheB基因表达的邻苯二酚2，3一双加氧酶活性比较弱，不很稳定，有待后续
工作中进一步完善。基因稳定性试验表明，户g基因能够在ZD4一1-fPg中稳定的存在，
由于ZD4一卜加g在480nm紫外光激发下能发出绿色荧光，因此该菌能够作为生物修复
的示踪菌，在污染环境生物修复中具有潜在的应用价值。
Three aromatic compounds degrading bacterial strains ZD 2, ZD 4-1 and ZD 4-3 were isolated from the sludge of pesticide manufacturing factory and the soil around the coal gas factory. All these isolates are Gram-negative, aerobic, non-spore-forming rods. Based on phenotypic characteristics and 16S rDNA sequence alignment, strains ZD 2, ZD 4-1 and ZD 4-3 were identified as Sphingobacterium multivorum, Comamonas testosteroni and Pseudomonas aeruginosa, respectively. The result of phylogenetic analysis showed that strain ZD 4-1 and ZD4-3 were positioned in two different subclusters. Cometabolism of pentachlorophenol (PCP) was studied by using Sphingobacterium multivorum ZD 2 to investigate the metabolism mechanism of PCP. The effect of PCP degradation with the phenol or glucose as growth substrates was compared. The results revealed that not only the PCP but also the phenol inhibited the bacterial cells and glucose was better than the phenol as a growth substrate for PCP cometabolism because there was no compet
itive inhibition between the glucose and PCP. Protein electrophoresis results showed that the unspecific enzymes for PCP degradation were induced by the non-growth substrate PCP itself when glucose was served as growth substrate. Gas chromatograph-Mass (GC-MS) analysis indicated that phenol was one of intermediate products during PCP cometabolism, suggesting that the dechlorination reaction increasingly happened even on the condition of aerobic biodegradation process.
To investigate the characteristic and biochemical mechanism about the biodegradation of aromatic compounds by bacteria, bacterial strains ZD 4-1 and ZD 4-3 were used to degrade phenol. The identification of the intermediates and the detection of the corresponding catabolic enzymes in crude extracts indicated that the strains ZD 4-1 and ZD 4-3 metabolized phenol via ortho-pathways and wefar-pathways, respectively. The results of induction experiment showed that the catechol dioxygenases, both catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O), were all inducible. Finally, the results of biodegradation and enzyme assays showed that the biodegradation efficiency of phenol by meta-Tpathways was higher than that by ortho-pathways but the former is in an inferiority
position in terms of adaptation to pH fluctuation and of growth ability on other aromatic compounds compared with the latter.
C23O is one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta-position of dihydroxylated aromatic substrates. C23O is able to catalyze the conversion of catechol to 2-hydroxymuconic semialdehyde (HMS). Based on curing experiment, PCR identification and Southern Hybridization, the C23O encoding gene pheB was localized on a 3.5 kb EcoRI/BamHI fragment and was then cloned from P. aeruginosa ZD 4-3 that was able to degrade both single and bicyclic compounds via a meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it has one open reading frame (ORF) encoding 307 amino acids, which showed a strong overall amino acid similarity to the known Gram-negative bacterial mesophilic C23Os. The heterogonous expression of the pheB gene in E. coll BL21 indicated that the activity of C23O was 3.5617U/mg protein and was higher that that of the C23O in wild bacterium.
In order to probe the relationship of the structure and function of C23O encoded by pheB gene, the alignment was performed by comparing the homology between the pheB deduced amino acids sequence and the other extradiol dioxygenases by the Bioinformatics software. The alignment analysis showed that 12 homology areas aroused during the whole sequences and these homology areas mainly distributed near the C-end and N-end. According to three-dimension crystal structure of the known extradiol dioxygenases, the amino acids Gly-31, His-157, Leu-184 were speculated to be the active sites of the C23O in this study. Moreover, there are evident differences between the extradiol dioxygenases which degrade single ring aromatic compounds, and those bicyclic
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