【作者】 李金霞；

【导师】 杜连祥；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 硕士

【摘要】 本论文研究了出发菌株地衣芽孢杆菌的生理及遗传特性，对它所产的耐高温α-淀粉酶的酶学性质作了初步分析。提取地衣芽孢杆菌的染色体DNA，设计合适引物，运用PCR法扩增得到了耐高温α-淀粉酶基因，并将其克隆在大肠杆菌中。运用重组PCR技术对耐高温α-淀粉酶基因进行定点突变。主要研究以下内容： 1．地衣芽孢杆菌020401所产耐高温α-淀粉酶的主要性质为：最适pH为6.5左右，最适作用温度为85℃，但在95℃时也能保持较高活性。 2．提取地衣芽孢杆菌020401的染色体DNA，设计引物，通过PCR扩增得到了1.9kb的基因片段。将其克隆在pUC19质粒上，并转化Escherichia coli JM109，运用蓝白选择及透明圈法筛选到了一株阳性克隆JM109／pUAM。 3．通过酶活测定及蛋白电泳分析证明JM109／pUAM菌株能分泌外源基因的表达产物即耐高温α-淀粉酶，目的蛋白大小约为60000左右。 4．对此基因片段进行测序，并将测序结果同已发表的地衣芽孢杆菌耐高温α-淀粉酶基因序列进行比对，同源性高达99％以上。 5．将该基因克隆在表达型载体pGEM-3Zf(+)上并转化E．coliJM109，同样得到了一株能产耐高温α-淀粉酶的阳性克隆JM109／pGAM，SDS-PAGE电泳证明其产生的目的蛋白大小同JM109／pUAM菌株大小相同，酶活测定表明菌株JM109／pGAM分泌目的蛋白的能力比JM109／pUAM强。 6．运用重组PCR技术对目的基因编码第134位氨基酸残基和第320位氨基酸残基的密码子分别进行了突变，将突变后的两种基因分别克隆在pGEM-3Zf(+)质粒上，转化E．coli JM109，通过蓝白选择及透明圈法筛选到了两株阳性克隆菌株JM109／pGAT1和JM109／pGAT2。更多还原

【Abstract】 The physiological and genetic characteristics of Bacillus licheniformis 020401 were studied, and the enzymic character of the heat-stable α-amylase produced by this strain was analyzed. The heat-stable α-amylase gene was amplified from B. licheniformis with PCR and cloned in Escherichia coli. The gene was mutated by recombinant PCR. The mainly researching contents were showed as below:1. The optimum pH and temperature of the heat-stable a-amylase produced by B. licheniformis 020401 was 6.5 and 85℃, separately, thought it could remain superior activity at 95℃.2. The chromosome DNA of B. lichenformis 020401 was extracted., The gene of 1.9kb was obtained by PCR with appropriate primers. Thegene was then connected with pUC19 plasmid and the recombinant plasmid was transformed into E. coli JM109. A positive clone JM109/pUAM was screened by the blue-white choose and halo method.3. It was proved that the JM109/pUAM could secret heat-stable α-amylase into the medium through measuring the activity of the a-amylase in the cell-free supernatant of the culture. The relative molecular weight of the a-amylase was about 60000 by SDS-PAGE.4. The sequence of the gene was menstruated and compared with the known sequence of the heat-stable a-amylase gene. The homology was above 99%.5. The aim gene was connected with pGEM-3Zf(+) plasmid and cloned into E. coli JM109, and as a result, a positive clone JM109/pGAM was obtained. It had the same protein producing ability as JM109/pUAM and the activity of the a-amylase was higher than that of the JM109/pUAM.6. The codes of the a-amylase gene were mutated at the amino acid 134 and 320, respectively. The two kinds of gene were separately connected with pGEM-3Zf(+) plasmid and transformed into E. coli JM109. Two positive clones, JM109/pGATl and JM109/pGAT2, were obtained.更多还原