《β-葡萄糖苷酶基因Syn-bgll在毕赤酵母中的高效表达及优化》-毕业论文（设计）.doc

PAGE 南京林业大学 本科毕业设计 题 目：β-葡萄糖苷酶基因Syn-bgll在毕赤酵母中的高效表达及优化 学 院：化学工程学院 专 业： 生物制药 学 号： 070204313 学生姓名： 指导教师： 职 称： 教授 二O一一 年 五 月 二十八 日 摘要 我们对以在毕赤酵母中有较高表达的黑曲霉中β-葡萄糖苷酶基因进行全基因优化，构建入表达载体pPICZαA-syn-bgll，然后在启动子的控制下在毕赤酵母中表达并获得重组毕赤酵母。 通过筛选获得的重组毕赤酵母，我们得到了酶活较高的8号转化子，并将其与原始菌株38号在同一水平下进行产酶对比；结果显示出：8号酶活比38号酶活有了明显提高。在第15天时，8号最高酶活可达27.9U/mL。 为了进一步提高8号菌株的酶活，我们对其进行二次电转来增加其拷贝子，从而来达到提高酶活的目的。通过实验结果显示，二次电转后的转化子B2酶活与8号相比，有了明显的提高。在第15天时B2最高酶活可达36.37U/mL，比8号提高了30.4%。 为了进一步优化B2产酶条件，我们对其进行响应面设计来确定其优化条件。利用Plackett-Burman设计法从12因素中筛选出对产酶水平有显著影响的三个因素,初始pH，甲醇添加量，装液量。根据Plackett-Burman实验分析结果，我们对得出的三个显著因素进行Box-Behnken中心组合实验设计，以β-葡萄糖苷酶的酶活为响应值建立二次回归方程,在分析各个因素的显著性和交互作用后,确定了重组毕赤酵母B2的最优培养基。 关键词：β-葡萄糖苷酶，全基因优化，黑曲霉，毕赤酵母，响应面 Abstract The β-glucosidase gene which cloned from Aspergillus niger had optimizated and constructed into the expression vector pPICZαA. And it successful expressed in Pichia pastoris under the control of promoter. By screening the recombinant Pichia pastoris, we obtained a higher activity of transformation 8# . Compared it with the original strain 38# at the same level of enzyme production; the results showed that the activities of 8# was more than 38#. On 15th day, the β-glucosidase activity by 8# was reached 27.9U/mL. To further enhance the activity of strain 8#, we carried 2th electric transformation to increase its transformants, in order to achieve more activity. The experiment results showed that the activity of transformant B2 by using 2th electric transformation, compared with the activity of 8#, was relatively higher. On 15th day, it’s β-glucosidase activity was reached 36.37U/mL. Comparing with the activity of 8# which was increased 30.4%. In order to further optimize the condition of enzyme production of B2, we used response surface methodology to determine the optimal conditions. Plackett-Burman design was used to evaluate the influence of twelve related factors. It was showed that there were three factors which was important for