目录
摘 要 III
关键词 III
Abstract IV
引言
引言 1
1 材料与方法 2
1.1 实验材料 2
1.1.1 菌株与质粒 2
1.1.2 化学试剂 2
1.1.3 试剂盒 2
1.1.4 培养基 2
1.2 实验方法 3
1.2.1 百菌清水解脱氯酶相关蛋白质系统进化分析 3
1.2.2 大肠杆菌感受态细胞制备 3
1.2.3 枯草芽孢杆菌感受态细胞制备 3
1.2.4 表达载体构建 3
1.2.5 大肠杆菌的转化和质粒提取 5
1.2.6 枯草芽孢杆菌的转化和质粒DNA的小量提取 5
1.2.7 酶的浓缩与纯化 5
1.2.8 酶活测定 5
1.2.9 重组枯草芽孢杆菌WBAPC3产Chd酶活发酵培养基的优化 6
2 结果与分析 7
2.1 百菌清水解脱氯酶在枯草芽孢杆菌中的表达 7
2.1.1 百菌清水解脱氯酶基因chd序列分析 7
2.1.2 百菌清水解脱氯酶（Chd）基因密码子优化 7
2.1.3 百菌清水解脱氯酶（Chd）相关蛋白质系统进化分析 8
2.1.4 百菌清水解脱氯酶（Chd）基因的PCR扩增 8
2.1.5 质粒酶切验证 9
2.1.6 枯草芽孢杆菌的转化子筛选 9
2.2 培养基中不同碳源及浓度对WBAPC3菌株产Chd酶活的影响 10
2.3 培养基中不同氮源及浓度对WBAPC3菌株产Chd酶活的影响 11
2.4 培养基中不同发酵温度及装液量对WBAPC3菌株产Chd酶活的影响 12
2.5 PLACKETTBURMAN实验 13
2.6 响应面法优化产Chd发酵培养基 15
3 结论与讨论 20
致谢 21
参考文献： 21
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表达
摘 要
枯草芽孢杆菌（Bacillus subtilis）广泛应用在工业发酵中，是一种重要工业酶制剂的生产菌种，具有可将目的基因表达产物分泌到胞外的独特优势。为提高百菌清水解脱氯酶chd基因的表达量，本课题首先对chd基因序列进行了分析，采用了密码子优化的策略，将优化后的基因导入并构建出一株枯草芽孢杆菌重组菌株WBAPC3，实现了chd基因的表达，同时进行了与Chd酶相关蛋白的系统进化分析。为了进一步优化重组枯草芽孢杆菌WBAPC3产Chd酶相关发酵条件，以达到提高酶产量的目的，设计了单因素试验，得到了在不同碳源及浓度、不同氮源及浓度、不同发酵温度和装液量条件下的产Chd酶活量，并运用PB试验和响应面分析法，得出在接种量9.35%（v/v）、搅拌速度197 rpm、MgSO4浓度0 g/L、KCl浓度6.97 g/L时，达到极值点，模型预测的最大响应值为14.1401 U/L，最适条件下进行的3组重复性试验，平均产Chd酶活为14.02 U/L，表明试验确定的模型条件可以用于预测实际值，对工业生产发酵具有重要的参考作用。
HIGHLEVEL EXPRESSION OF CHLOROTHALONIL HYDROLYTIC DECHLORINASE (Chd) IN Bacillus subtilis
ABSTRACT
Bacillus subtilis as an important industrial enzyme production strain, is widely used in industrial fermentation. Bacillus subtilis has the unique advantage of secreting the expressed products of the target gene outside the cell. In order to improve the expression of chd gene of Chlorothalonil hydrolyzed dechlorinase, firstly, the sequence of chd gene was analyzed and the strategy of codon optimization was adopted. The optimized gene was introduced into Bacillus subtilis and a recombinant strain WBAPC3 was constructed to express chd gene. At the same time, the phylogenetic analysis of proteins related to Chd enzyme was carried out. In order to optimize the fermentation conditions related to the production of Chd enzyme by recombinant Bacillus subtilis WBAPC3, a single factor experiment was designed to obtain the activity of Chd enzyme under the conditions of different carbon source and concentration, different nitrogen source and concentration, different fermentation temperature and liquid volume. PB test and response surface analysis were used. The results showed that the maximum value was reached when the inoculation size was 9.35% (v/v), the stirring speed was 197 rpm, the MgSO4 concentration was 0 g/L and the KCl concentration was 6.97 g/L. The maximum response value predicted by the model was 14.1401 U/L. Under the optimum conditions, the average Chd enzyme activity was 14.02 U/L, indicating that the model conditions determined in the experiment can be used to predict the actual value. This is an important reference for industrial production and fermentation.

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