【作者】 孙峥；

【导师】 章力建； 许雷；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 漆酶是一种含多个铜原子的多酚氧化酶，能催化氧化多种芳香族化合物，在木质素矿化降解、腐殖质形成等过程中发挥重要作用。其应用主要集中在以下几方面：参与有机合成，生物检测，有毒化合物的消除，工业废水处理，纸浆的生物漂白等。本试验从北京昌平某羊场采集的堆肥样品中分离筛选出一株生长快速，菌落规则，传代稳定，具有漆酶活性的细菌菌株，并对该菌株的生理生化性质进行了初步研究，此外，通过构建基因组文库，对该菌株中的漆酶编码基因进行了克隆。具体实验结果如下：1．漆酶活性测定表明：该菌株产胞外漆酶，漆酶活性最高可达32.3U／L。2．Biolog鉴定和16S rDNA序列分析结果表明：该菌为Pseudomonas sp．(假单胞菌)属菌株，革兰氏阴性菌。3．该菌株的最适生长温度为27℃；最适生长pH值为7.0，生长pH范围为6.0～8.0。菌株可耐受2％的盐离子浓度，对青霉素有抗性。4．以pGEM-3Z为载体、E．coli JM109为受体菌，构建了该菌株的基因组文库，文库库容达到10~4，插入片段长度约为2～7kb。以漆酶的产生为筛选标志，对构建的基因组文库进行筛选，得到了具有漆酶活性的重组菌，经测序得到2458bp的核苷酸序列。阅读框架分析表明：该插入片段中含有一个5’端完整、长1193bp、编码397aa的ORF，在ORF上游约-50bp～-100bp的区域为启动子区；前13个氨基酸组成信号肽，信号肽切点位于ATG下游的第14个氨基酸Thr上。该序列已在GenBank注册，注册号EF067317。5．漆酶活性测定表明：重组菌株的漆酶活力为20.4 U／L。现有文献中尚未发现有关假单胞菌具有漆酶活性的报道，本研究首次分离到具有漆酶活性的假单胞菌，并克隆到具有活性漆酶编码能力的漆酶基因片段，具有原创性。更多还原

【Abstract】 Laccase, capable of oxidizing a wide range of aromatic compounds, is a type of multicopper polyphenol oxidases.The enzyme plays an important role in the biodegradating of lignin and formatting of humus in composting. Recently, laccase is widely applied in organic syntheses, bio-detection, industrtial wastewater treatment and so on.In this experiment, a bacterial strain with laccase activity was isolated from the dunghill samples. The physicochemical properties of this srain were studied. In addition, the laccase gene has been cloned by constructing a genomic library. The detail results as followed.1. Laccase activity of L-1 strain was 32.3U/L respectively.2. By Biolog test and 16S rDNA sequence analysis, the L-1 strain was identified as Pseudomonas sp., it’s a gram-negative bacteria.3. The optimum temperature of L-1 strain is 27℃, and the optimum pH is 7.0. Additionally, its growth pH range was 6.0 to 8.0. It confers salt-tolerance of 2%, besides it can resist Penicillin.4. In this experiment, we constructed the genomic library of the bacteria, with the E.coli JM109 as the receptor strain, and pGEM-3Z as the vector. We got the genomic library with the insert fragments among 2~7kb, and the capacity is 104. A recombinant bacterial strain with laccase activity was screened.Then sequenced and analysed, the results showed that the full-length insert fragment composed 2458bp, including an integrant open reading frame which is 1193bp at length, composed 397 amino acid, with 13-amino-acid signal petide. The ORF was 5’ integrity, 3’ missed, and its expected promoter sequence was located at position -50 to -100. The sequence fragment has been submitted to the GenBank, and the accession number is EF067317.5. The laccase activity of recombinant strain is 20.4 U/L.At present, there is no report about the laccase activity of Pseudomonas sp., this is the first time that the gene encoding an active laccase has been cloned from Pseudomona sp.更多还原