【作者】 艾海新；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境工程， 2008， 博士

【摘要】 硝基苯废水成分复杂,常含有大量对微生物有抑制作用的盐。基于此,本论文驯化、筛选耐盐硝基苯降解菌,对其进行生理生化鉴定,研究其耐盐和降解硝基苯特性。在此基础上,对其降解硝基苯代谢基因进行定位,并进一步利用现代分子生物学技术克隆到部分基因并对其进行了生物信息学分析。从活性污泥中驯化分离到2株耐盐硝基苯降解菌,通过形态观察、生理生化分析、G+C含量分析和16S rDNA分析分别鉴定为Streptomyces sp.DUT_AHX和Micrococcussp.DUT_AHX,其16S rDNA序列的GenBank登录序列号分别为DQ409080和DQ409081。Streptomyces sp.DUT_AHX的最适生长与降解条件为:温度30℃,pH 7.0,转速150r/min,菌株在唯一碳源培养基中可耐受的最大硝基苯浓度为900 mg/L。Micrococcus sp.DUT_AHX的最适生长与降解条件为:温度37℃,pH 7.0,转速150 r/min,菌株在唯一碳源培养基中可耐受的最大硝基苯浓度为600 mg/L。2株菌在以硝基苯为唯一碳、氮和能源的无机盐培养基中生长和降解硝基苯过程中均释放出NH₄⁺,但未检测到NO₂^-。在80 h内2株菌株对硝基苯的降解率分别为98.8%和97.6%,TOC去除率为99.2%和98.1%,表明硝基苯最终被矿化为二氧化碳和水。2株菌的粗酶主要以2-氨基酚1,6-双加氧酶活性为主。这2株菌降解硝基苯的途径可能都是好氧部分还原途径。Streptomyces sp.DUT_AHX是一株中度耐盐细菌。在盐胁迫下某些相容性溶质如甘氨酸、脯氨酸、谷氨酸、甜菜碱、四氢嘧啶都能更好的促进其生长,其中甘氨酸的效果最明显,四氢嘧啶的效果最差。利用SDS-PAGE和非变性梯度PAGE电泳分析了盐胁迫或硝基苯诱导下Streptomyces sp.DUT_AHX蛋白质的合成变化,其中一些蛋白质的合成被抑制;一些蛋白质的合成被增强:一些起主要调节作用的盐或硝基苯诱导蛋白被重新合成。将非变性梯度PAGE凝胶上的大小为141 kDa的硝基苯诱导蛋白条带切胶电洗脱回收,其具有2-氨基酚1,6-双加氧酶活性,其活性大小为5.825μmol/min/mg protein,该蛋白是Streptomyces sp.DUT_AHX降解硝基苯代谢途径中的2-氨基酚1,6-双加氧酶。从Streptomyces sp.DUT_AHX提取到一大小约为17 kb的质粒pSNB1。采用提高生长温度和SDS法相结合消除质粒,筛选到一质粒消除菌株AHX-4,失去了氨苄青霉素抗性和降解硝基苯的能力。将质粒pSNB1经CaCl₂热激转化法进入大肠杆菌JM109感受态细胞,筛选到一个可在以硝基苯为唯一碳源的无机盐平板上生长的转化大肠杆菌阳性子AHX-JM109,其降解硝基苯的能力大大降低。质粒pSNB1与Streptomyces sp.DUT_AHX的抗性基因和降解硝基苯的基因有关,而与耐盐基因无关。以质粒pSNB1为模板和RedF、RedR为引物的简并降落PCR扩增到一大小为465 bp的基因片段,氨基酸序列检索表明,其与Marine actinobacterium PHSC20C1的YHS域蛋白的同源性较高;设计特异性引物进行盒式连接介导PCR,拼接得到大小为2026 bp的序列,开放阅读框分析发现一个编码126个氨基酸的ORF,属于YHS域蛋白,其代表的基因可能是Streptomyces sp.DUT_AHX的硝基苯硝基还原酶基因PPH_AHX,其GenBank中登录的序列号为EF998874。生物信息学分析表明,pPH_AHX基因编码的蛋白理论等电点为4.8,理论分子量为13.49089 kDa,为一位于细胞质的弱酸性、水溶性球状蛋白。通过不同程序预测其二级结构和三级结构,并构建了该蛋白部分三维结构模型。更多还原

【Abstract】 The purpose of this dissertation is to investigate the characteristics and the partial gene of novel salt-tolerant bacteria capable of utilizing nitrobenzene （NB） as the sole carbon, nitrogen and energy sources. The exploitation of salt-tolerant bacteria would be a remarkable improvement in NB bioremediation and wastewater treatment in high salinity. The physiological and biochemical tests and 16S ribosomal DNA （rDNA） sequence analysis were carried out. The characteristics of tolerating salt and degrading NB of Streptomyces sp. DUT_AHX and Micrococcus sp. DUT_AHX were also investigated. Meanwhile, catabolic genes of degrading NB were localized. The partial gene was cloned by modern molecular biological techniques and then was analyzed by bioinformatics techniques.Two novel salt-tolerant bacteria Streptomyces sp. DUT_AHX and Micrococcus sp. DUT_AHX, which were isolated from the sludge and could utilize NB as the sole carbon, nitrogen and energy sources, were identified on the basis of physiological and biochemical tests and 16S rDNA sequence analysis. The 16S rDNA sequences of Streptomyces sp. DUT_AHX and Micrococcus sp. DUT_AHX were submitted to GenBank with the accession number DQ409080 and DQ409081, respectively.The optimal degradation and growth conditions of Streptomyces sp. DUT_AHX are as follows: temperature 30℃, pH 7.0 and shaking speed 150 r/min. It can grow in the presence of NB up to 900 mg/L in mineral salts basal （MSB） medium. The optimal degradation and growth conditions of Micrococcus sp. DUT_AHX are as follows: temperature 37℃, pH 7.0 and shaking speed 150 r/min. It can grow in the presence of NB up to 600 mg/L in MSB medium. Besides, NB-grown cells in MSB medium can degrade NB with the concomitant release of ammonia. The NB degradation rate of strain 1# and strain 2# were 98.8% and 97.6% in 80 h and TOC removing rate were 99.2% and 98.1%. Thus, NB is mineralized to CO₂ and H₂O. The enzyme activity tests show that crude extracts mainly contain 2-aminophenol 1,6-dioxygenase activity. Thus, they may involve a partial reductive pathway of degrading nitrobenzene.Streptomyces sp. DUT_AHX can tolerate moderately NaCl but does not require NaCl for growth. Hence, it is not halophilic but halotolerant. The exogenously added osmoprotectants such as glycin, glutamic acid, proline, betaine and ectoine can improve growth of Streptomyces sp. DUT_AHX in the presence of 10% （w/v） NaCl. The proteins induced by salinity stress or NB were analyzed by native-gradient polyacrylamide gel electrophoresis （PAGE） and sodium dodecyl sulfate （SDS）-PAGE. There were three prominent types of modifications, which several proteins were declined, certain proteins were enhanced and some proteins were induced de novo. In NB-induced proteins de novo, 141 kDa protein on the native-gradient PAGE gel was excised and electroeluted. Furthermore, enzyme tests exhibit the 2-aminophenol 1,6-dioxygenase activity of purified 141 kDa protein is 5.825μmol/min/mg protein, which is 11-fold that of the cell-free extracts. Thus, 141 kDa protein is the 2-aminophenol 1,6-dioxygenase of Streptomyces sp. DUT_AHX.A 17-kb plasmid was detected in Streptomyces sp. DUT_AHX and designated pSNB1. The use of elevating growth temperature together with SDS was effective in curing the plasmid pSNB1. A cured derivative designated AHX-4 lost NB degradability and ampicillin resistance. Moreover, pSNB1 was successfully transformed into E. coli JM109 competent cells by calcium chloride method and one transformant AHX-JM109 capable of degrading NB was obtained. As a result, some NB catabolic genes and antibiotic resistance genes are plasmid-mediated in Streptomyces sp. DUT_AHX.Moreover, the plasmid DNA was amplified with degenerate primers by touchdown polymerase chain reaction （PCR） and an expected size fragment （465 bp） was generated. The Blast results reveal that the gene encoding a 155 amino acid polypeptide is 33% to 76% identical to YHS domain protein. The sequences amplified by cassette-ligation-mediated PCR were spliced and the sequence is 2026 bp. The open reading frames （ORFs） in the sequence were analyzed by ORF Finder program. The ORF encoding 126 amino acid polypeptide is identical to YHS domain protein. The ORF designated pPH_AHX may be the gene encoding NB nitroreductase of Streptomyces sp. DUT_AHX. The sequence of pPH_AHX gene was submitted to GenBank with the accession number EF998874. The pPH_AHX gene was analyzed by bioinformatics techniques. It shows that the isoelectric point of the protein encoded by pPH_AHX gene is 4.8 and the theoretic molecular weight is 13.49089 kDa. The protein is acidic, soluble and spherical in cytoplasm. Furthermore, the partial second and tertiary structure of the protein were predicated and three-dimensional structure homologous model was constructed.更多还原