農(nóng)藥污染土壤中漆酶基因的高通量篩選與克隆
發(fā)布時間：2018-12-19 07:20
【摘要】：漆酶(EC 1.10.3.2)是自然界廣泛分布的一種多銅氧化酶,屬于藍(lán)色多銅氧化酶家族中的一個成員,它能氧化酚類及衍生物、芳胺類及衍生物、羥酸類及衍生物、甾體激素類、生物色素類及其它非酚類的相關(guān)底物等。目前報道,漆酶廣泛的存在于細(xì)菌中,相比真菌漆酶,細(xì)菌漆酶具有不需糖基化、熱力學(xué)穩(wěn)定性好,酶活最適pH廣泛等更多的優(yōu)勢。從農(nóng)藥污染的土壤中篩選具有熱穩(wěn)定性良好的細(xì)菌漆酶,具有很重要的工業(yè)價值。本研究從湖北蘄春農(nóng)藥廠的一級好氧池中采集土壤樣品,首先提取土壤樣品的宏基因組DNA,根據(jù)微生物漆酶的保守區(qū)設(shè)計引物,PCR擴(kuò)增漆酶基因片段,通過測序分析土壤樣品中的漆酶多樣性,在測序的100個陽性克隆子中,有62條不同的漆酶基因序列,證明漆酶多樣性較好。進(jìn)而展開土壤宏基因組Fosmid文庫的構(gòu)建工作。通過建庫,共獲得6000余個克隆子。從所構(gòu)建的Fosmid文庫中篩選不同的漆酶基因,目前共篩選得到6個漆酶基因(lac2A3、lac2E6、lac13H9、lac13B22、lac15A5、lac15P1),通過Signal P4.1 Server軟件對6個漆酶的信號肽進(jìn)行分析,發(fā)現(xiàn)漆酶基因lac2A3、lac13H9、lac15A5均分別含有36個,17個,30個氨基酸的信號肽序列。在分別去掉信號肽的情況下,與其它3個沒有信號肽的漆酶基因一起分別與載體pET-22b和pET-30a構(gòu)建重組表達(dá)載體,轉(zhuǎn)化大腸桿菌Transseta(DE3)菌株,最后發(fā)現(xiàn)僅重組酶Lac13H9可溶性表達(dá),其他均形成包涵體蛋白而沒有活性。對漆酶基因lac13H9進(jìn)行信息分析,其全長為1389 bp,編碼462個氨基酸,推斷其理論分子量為50 kDa,用Signal p 4.1軟件分析,其含有一個17個氨基酸的信號肽,與NCBI蛋白數(shù)據(jù)庫比對結(jié)果表明,與來源于Myxococcus stipitatus的多銅氧化酶序列相似性最高為67%,其次與來源于Sorangium cellulosum的多銅氧化酶序列相似性為61%,表明這是一個新的漆酶基因。將其在大腸桿菌Transseta(DE3)中通過微好氧發(fā)酵進(jìn)行異源表達(dá),并通過Ni柱親和層析純化重組酶lac13H9。獲得的重組漆酶Lac13H9具有良好的酶學(xué)性質(zhì),其最適溫度為40℃,最適pH為6.0,且具有較好的熱穩(wěn)定性,在50℃下保溫90 min還有60%的剩余酶活力,同時發(fā)現(xiàn)低濃度的Fe2+促進(jìn)漆酶酶活力,高濃度則抑制酶活力。良好的酶學(xué)性質(zhì)為該酶的應(yīng)用奠定了基礎(chǔ)。
[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，
本文編號：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，
本文編號：2386594
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