【作者】 程夕冉；

【导师】 武波；

【作者基本信息】 广西大学 ， 微生物， 2007， 硕士

【摘要】 本工作从土壤中筛选到一株能利用苯甲酸钠产邻苯二酚的假单胞菌株B3-1。通过对菌株B3-1在不同底物浓度、温度和pH条件下产邻苯二酚条件的优化，邻苯二酚产量最高能达到1.57mg／mL，其最适生长条件为32℃、pH 6.0～7.0，产邻苯二酚的最佳底物浓度为3mg／mL。以假单胞菌B3-1为出发菌株，进行了固定化细胞生产邻苯二酚的初步研究。通过明胶、海藻酸钠和聚乙烯醇三种固定化材料的比较，最终确定了使用海藻酸钠作为固定化载体。通过正交实验，固定化的最佳条件为：菌体量0.3 g／mL；氯化钙浓度为4％；海藻酸钠浓度为3％；钙化时间为4小时。用柯斯质粒pWEB：：TNC为载体构建了Pseudomonas sp．B3-1含约20000个克隆的基因组文库，随机挑取其中的14个克隆提取质粒进行BamHI酶切分析，结果显示所有的质粒都含有插入片段，最大为52kb，最小为31kb，平均大小为41.5 kb，14个质粒的酶切带型都不相同，说明文库克隆的外源DNA片段随机性比较大。通过活性筛选得到含编码邻苯二酚合成基因的阳性克隆45个。从阳性克隆Ben19中得到苯甲酸双加氧酶基因benABCD，BlastX表明该序列与来自于Pseudomonas entomophila str．L48(GenBank索引号：CT573326)的苯甲酸双加氧酶ben基因簇有91％的同源性。benA具有一个1329bp的ORF，可编码含442个氨基酸的蛋白质，编码产物与来自假单胞菌的苯甲酸钠双加氧酶BenA(GenBank索引号AAX47023.1)具有96％一致性和99％相似性。benB具有一个486bp的ORF，编码含161个氨基酸的蛋白质，编码产物与来自Pseudomonas entomophila str．L48的苯甲酸钠双加氧酶BenB(GenBank索引号AAX47024.1)具有91％一致性和97％相似性。benC具有一个1011bp的ORF，编码含336个氨基酸的蛋白质，编码产物与来自假单胞菌的苯甲酸钠双加氧酶BenC(GenBank索引号AAX47025.1)具有93％一致性和98％相似性。benD具有一个762bp的ORF，编码含253个氨基酸的蛋白质，编码产物与来自假单胞菌的苯甲酸二醇脱氢酶BenD(GenBank索引号AAK52290.1)具有79％一致性和88％相似性。更多还原

【Abstract】 A strain was isolated from soil for utilizing sodium benzoate to producecatechol, and identified as Pseudomonas sp. B3-1. The maximum yield ofcatechol reached 1.57 mg/ml after optimizing sunstrate contration, reactiontemperature and pH. The optimal substrate concentration was 3 mg/ml. Theoptimal temperature and pH for growth were 32℃and 6.0～7.0, respectively.Immobilized Pseudomonas sp. B3-1 cells were prepared by gelatin,sodium alginate and polyvinyl alcohol as supports, and applied for producingcatechol. Sodium alginate was Choosen for further study. The optimumimmobilized conditions were 3% sodium alginate, 4% （w/v）CaCl₂, wet cellweight 0.3g/ml, and 4h for immobilization.A DNA library of Pseudomonas sp. B3-1 containing about 20000 cloneswas constructed by cosmid vector pWEB::TNC. 14 clones were randomlychosen from the library and extracted their plasmids. The plasmids weredigested with BamHI and analyzed by agarose gel electrophoresis. The result showed that each of them harbored different foreign DNA fragments. And thelarge insert DNA fragment was 52 kb and the smallest was 31kb. The averagesize of the insert DNA fragment was 41.5 kb. The restriction analysis of 14plasmids from library revealed that the cloned DNA fragments were random andthe library had a high quanlity.45 positive clones were obtained from the library using function-basedscreening strategy of catechol. Benzoate genes benABCD were cloned frompositive clone Ben19. The ORF of gene benA was 1329 bp, which encodeda production of 442 amino acids, shared 96% identities and 99% homology toBenA of Pseudomonas entomophila str. L48 （accession No. AAX47023.1.）; ThebenB harboring a 486 bp ORF, which encoded a protein of 161 amino acids,sharing 91% identities and 97% homology to BenB of Pseudomonasentomophila str. L48（GenBank accession No. AAX47024.1）. The benCharboring a 1011 bp ORF, which encoded a protein of 336 amino acids,sharing 93% identities and 98% homology to BenC of Pseudomonasentomophila str. L48（GenBank accession No. AAX47025.1）. The benDharboring a 762 bp ORF, which encoded a protein of 253 amino acids, sharing79% identities and 88% homology to BenC of Pseudomonas putida （GenBankaccession No. AAK52290.1.）.更多还原