农药污染土壤中漆酶基因的高通量筛选与克隆
发布时间：2018-12-19 07:20
【摘要】：漆酶(EC 1.10.3.2)是自然界广泛分布的一种多铜氧化酶,属于蓝色多铜氧化酶家族中的一个成员,它能氧化酚类及衍生物、芳胺类及衍生物、羟酸类及衍生物、甾体激素类、生物色素类及其它非酚类的相关底物等。目前报道,漆酶广泛的存在于细菌中,相比真菌漆酶,细菌漆酶具有不需糖基化、热力学稳定性好,酶活最适pH广泛等更多的优势。从农药污染的土壤中筛选具有热稳定性良好的细菌漆酶,具有很重要的工业价值。本研究从湖北蕲春农药厂的一级好氧池中采集土壤样品,首先提取土壤样品的宏基因组DNA,根据微生物漆酶的保守区设计引物,PCR扩增漆酶基因片段,通过测序分析土壤样品中的漆酶多样性,在测序的100个阳性克隆子中,有62条不同的漆酶基因序列,证明漆酶多样性较好。进而展开土壤宏基因组Fosmid文库的构建工作。通过建库,共获得6000余个克隆子。从所构建的Fosmid文库中筛选不同的漆酶基因,目前共筛选得到6个漆酶基因(lac2A3、lac2E6、lac13H9、lac13B22、lac15A5、lac15P1),通过Signal P4.1 Server软件对6个漆酶的信号肽进行分析,发现漆酶基因lac2A3、lac13H9、lac15A5均分别含有36个,17个,30个氨基酸的信号肽序列。在分别去掉信号肽的情况下,与其它3个没有信号肽的漆酶基因一起分别与载体pET-22b和pET-30a构建重组表达载体,转化大肠杆菌Transseta(DE3)菌株,最后发现仅重组酶Lac13H9可溶性表达,其他均形成包涵体蛋白而没有活性。对漆酶基因lac13H9进行信息分析,其全长为1389 bp,编码462个氨基酸,推断其理论分子量为50 kDa,用Signal p 4.1软件分析,其含有一个17个氨基酸的信号肽,与NCBI蛋白数据库比对结果表明,与来源于Myxococcus stipitatus的多铜氧化酶序列相似性最高为67%,其次与来源于Sorangium cellulosum的多铜氧化酶序列相似性为61%,表明这是一个新的漆酶基因。将其在大肠杆菌Transseta(DE3)中通过微好氧发酵进行异源表达,并通过Ni柱亲和层析纯化重组酶lac13H9。获得的重组漆酶Lac13H9具有良好的酶学性质,其最适温度为40℃,最适pH为6.0,且具有较好的热稳定性,在50℃下保温90 min还有60%的剩余酶活力,同时发现低浓度的Fe2+促进漆酶酶活力,高浓度则抑制酶活力。良好的酶学性质为该酶的应用奠定了基础。
[Abstract]:Laccase (EC 1.10.3.2) is a polycopper oxidase widely distributed in nature. It belongs to the blue polycopper oxidase family. It can oxidize phenols and derivatives, aromatic amines and derivatives, hydroxyl acids and derivatives. Steroid hormones, biopigments and other non-phenolic related substrates. It has been reported that laccase is widely present in bacteria. Compared with fungal laccase, laccase has many advantages, such as no glycosylation, good thermodynamic stability and wide range of pH. Screening bacterial laccase with good thermal stability from pesticide contaminated soil is of great industrial value. In this study, soil samples were collected from the primary aerobic pool of Qichun Agricultural Pharmaceutical Factory in Hubei Province. The macro genomic DNA, of soil samples was first extracted and primers were designed according to the conservative region of microbial laccase, and the laccase gene fragments were amplified by PCR. The laccase diversity in soil samples was analyzed by sequencing. There were 62 different laccase gene sequences in 100 positive clones, which proved that laccase diversity was better. Then the construction of soil macro genomic Fosmid library was carried out. More than 6000 clones were obtained by building the library. Six laccase genes (lac2A3,lac2E6,lac13H9,lac13B22,lac15A5,lac15P1) were screened from the constructed Fosmid library, and six laccase genes (lac2A3,lac2E6,lac13H9,lac13B22,lac15A5,lac15P1) were obtained. The signal peptides of laccase were analyzed by Signal P4.1 Server software, and the laccase gene lac2A3, was found. Lac13H9,lac15A5 contains 36, 17 and 30 amino acid signal peptide sequences respectively. When the signal peptides were removed separately, the recombinant expression vectors were constructed with the vector pET-22b and pET-30a together with the other three laccase genes without signal peptide, respectively. The recombinant expression vector was transformed into E. coli Transseta (DE3) strain. Finally, only the soluble expression of recombinant enzyme Lac13H9 was found, and the others formed inclusion body protein without activity. The information of laccase gene lac13H9 was analyzed. The total length of laccase gene was 1389 bp, encoding 462 amino acids. It was deduced that the theoretical molecular weight of laccase gene was 50 kDa,. It was analyzed by Signal p4.1 software. It contained a signal peptide of 17 amino acids. The results of comparison with NCBI protein database showed that the similarity of polycopper oxidase sequence with Myxococcus stipitatus was the highest, followed with that of polycopper oxidase from Sorangium cellulosum was 61%, which indicated that it was a new laccase gene. It was expressed in E. coli Transseta (DE3) by microaerobic fermentation and purified by Ni affinity chromatography. The recombinant laccase Lac13H9 has good enzymatic properties, the optimum temperature is 40 鈩，
本文编号：2386594
[Abstract]:Laccase (EC 1.10.3.2) is a polycopper oxidase widely distributed in nature. It belongs to the blue polycopper oxidase family. It can oxidize phenols and derivatives, aromatic amines and derivatives, hydroxyl acids and derivatives. Steroid hormones, biopigments and other non-phenolic related substrates. It has been reported that laccase is widely present in bacteria. Compared with fungal laccase, laccase has many advantages, such as no glycosylation, good thermodynamic stability and wide range of pH. Screening bacterial laccase with good thermal stability from pesticide contaminated soil is of great industrial value. In this study, soil samples were collected from the primary aerobic pool of Qichun Agricultural Pharmaceutical Factory in Hubei Province. The macro genomic DNA, of soil samples was first extracted and primers were designed according to the conservative region of microbial laccase, and the laccase gene fragments were amplified by PCR. The laccase diversity in soil samples was analyzed by sequencing. There were 62 different laccase gene sequences in 100 positive clones, which proved that laccase diversity was better. Then the construction of soil macro genomic Fosmid library was carried out. More than 6000 clones were obtained by building the library. Six laccase genes (lac2A3,lac2E6,lac13H9,lac13B22,lac15A5,lac15P1) were screened from the constructed Fosmid library, and six laccase genes (lac2A3,lac2E6,lac13H9,lac13B22,lac15A5,lac15P1) were obtained. The signal peptides of laccase were analyzed by Signal P4.1 Server software, and the laccase gene lac2A3, was found. Lac13H9,lac15A5 contains 36, 17 and 30 amino acid signal peptide sequences respectively. When the signal peptides were removed separately, the recombinant expression vectors were constructed with the vector pET-22b and pET-30a together with the other three laccase genes without signal peptide, respectively. The recombinant expression vector was transformed into E. coli Transseta (DE3) strain. Finally, only the soluble expression of recombinant enzyme Lac13H9 was found, and the others formed inclusion body protein without activity. The information of laccase gene lac13H9 was analyzed. The total length of laccase gene was 1389 bp, encoding 462 amino acids. It was deduced that the theoretical molecular weight of laccase gene was 50 kDa,. It was analyzed by Signal p4.1 software. It contained a signal peptide of 17 amino acids. The results of comparison with NCBI protein database showed that the similarity of polycopper oxidase sequence with Myxococcus stipitatus was the highest, followed with that of polycopper oxidase from Sorangium cellulosum was 61%, which indicated that it was a new laccase gene. It was expressed in E. coli Transseta (DE3) by microaerobic fermentation and purified by Ni affinity chromatography. The recombinant laccase Lac13H9 has good enzymatic properties, the optimum temperature is 40 鈩，
本文编号：2386594
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