【作者】 潘皎；

【导师】 张义正；

【作者基本信息】 四川大学 ， 遗传学， 2004， 博士

【摘要】 短小芽孢杆菌（Bacillus pumilus）UN-31-C-42是一株从成都市尘活垃圾中分离出的菌株经过多次诱变后获得的碱性蛋白酶高产菌株。该菌株发酵液不但脱毛效果很好，而且胶原水解活性很低，因此在皮革工业中将有很好的应用前景。从该菌发酵液中已经分离纯化出能够独立完成脱毛过程的碱性蛋白酶DHAP，并且克隆到相应的蛋白酶基因AP。为了进一步证明AP基因编码的蛋白酶能否单独完成生皮的脱毛过程和建立能产生脱毛碱性蛋白酶的基因工程菌，本论文开展了以下4个方面的研究工作：建立枯草杆菌转化系统，克隆芽孢杆菌的强基因启动子，构建AP基因表达质粒，以及融合的AP基因在枯草杆菌WB600中的异源表达。 在枯草杆菌的转化系统的研究中，首先完成了短小芽孢杆菌UN-31-C-42和枯草杆菌WB600的生长曲线和它们对抗生素的敏感性的测定，结果表明，它们对卡那霉素都敏感，因此可以选择卡那霉素抗性基因作为这2种菌转化时的标记基因；短小芽孢杆菌对氯霉素敏感，而枯草杆菌WB600对氯霉素的抗性介于10～25μg／mL之间。从穿梭质粒载体pSUGV4中克隆到全长卡那霉素抗性基因和测定了它的序列，该序列与金黄色葡萄球菌（Staphylococcus aureus）的质粒pUB110中的卡那霉素抗性基因序列同源性高达100％。比较了3种转化枯草芽孢杆菌WB600方法的效果，发现使用感受态转化法时，若将OD₅₆₀为1.0的细菌培养物转入MD培养基进行饥饿培养，其转化率最高，可达到1.4×10³个／μg DNA；四川大学博士学位论文短小芽抱杆菌的碱性蛋白酶基因的表达研究利用原生质体转化时，原生质体形成条件以0.5 mg/mL溶菌酶用量，37℃下摇床温和处理时间lh为最佳，转化率是1.0x10，个/陀DNA:当用电激法时，枯草杆菌WB600培养至OD560为0.8时收获细胞，用SHMG缓冲液洗涤和悬浮细胞，取1卜L（50ng/林L）质粒pSUGv4与60林L细胞混合，在场强为16KV/cm，电阻200。，电容25叮条件下进行电脉冲，虽然可获得最高转化率，但转化率仅为2.4xl0，个/陀DNA。 在基因启动子克隆的研究中，首先利用启动子探针型载体pSUPv4直接在大肠杆菌中克隆芽抱杆菌的强基因启动子片段。从短小芽抱杆菌UN一31一C一42中获得5个较强启动转录功能的DNA片段（命名为Bp21、Bp38、Bp53，Bp57和Bp70）:从枯草杆菌WB600中分离到3个较强的基因启动子片段（命名为BsZ，Bs4和Bss）。DNA序列测定和同源性分析表明，它们都是新的DNA序列，并且具有较典型的原核生物基因启动子的保守序列。然后选择2个卡那霉素抗性最高的基因启动子片段进行了功能鉴定，短小芽抱杆菌的Bp53片段在大肠杆菌和枯草杆菌wB600中都能有效地启动短小芽抱杆菌碱性蛋白酶基因的表达;枯草杆菌的BsZ片段可以在大肠杆菌中高效地启动碱性蛋白酶基因的表达，也能在枯草杆菌中启动卡那霉素抗性基因的表达。此外，还从枯草杆菌WB600中克隆到组成型表达的双启动子P43和蔗糖诱导的果聚糖蔗糖酶基因SacB的启动子和信号肤，并测定了它们的序列，与己报道的枯草杆菌相应序列几乎完全相同，但在功能鉴定时，在枯草杆菌中未获得融合基因的表达。 在碱性蛋白酶基因表达质粒的构建工作中，先用PCR方法从短小芽抱杆菌UN一31一C一42总DNA中克隆到1 80bp的BP53启动子片段和1300bP的碱性蛋白酶AP基因的编码区序列（包括信号肤，前肤，成熟肤）和终止子序列。然后将此2个片段重组成融合基因（命名为 BpAP），融合处的序列经测定后证明与预期序列完全一致。将融合基因插入到大肠杆菌一枯草杆菌穿梭载体PsuGv4的多克隆位点区中，成功构建了碱性蛋白酶基因的表达质粒PsU一BpAP;经PCR验证和限制酶酶切分析，证明表达质粒PSU一BpAp与预期结果一致。 在BpAP基因的表达研究中，将构建的表达质粒psu一BpAp转入到枯草杆菌wB600中，得到转化子psU一BpAp朋B600，转化子在没有选择压四川大学博士学位论文短小芽饱杆菌的碱性蛋白酶基因的表达研究力的条件下，经过3次传代连续培养72h后，质粒发生丢失。转化子psu一BpAp/WB600能够在牛奶平板上产生透明的水解圈，其发酵上清液的SDS一PAGE分析能清晰的观察到32KD的表达产物，这些结果都表明了融合后的碱性蛋白酶基因在枯草杆菌中得到了表达。对转化子pSU一BpAp/wB600的发酵上清液进行酶活测定，结果表明，该菌株在发酵20h时开始产酶，在48h时达到高峰，但是该转化子产酶量很低，仅为32U/mL。将转化子的48h发酵上清液进行生皮脱毛实验，结果表明，发酵上清液有很好的脱毛效果，对皮革胶原没有造成损伤。取短小芽饱杆菌UN·31一C一42（作为正对照）和转化子pSU一BpAp/WB600的48h发酵上清液进行单位生皮面积的脱毛效果比较，结果表明，当它们在单位生皮面积上的酶量一致时，它们的脱毛效果也是一致的。该结果进一步说明，来自短小芽抱杆菌的碱性蛋白酶可以单独完成脱毛过程。对PSU一BpAP/wB600发酵条件进行了优化，酶活提高了4倍，可达到120U/mL。比较了3种培养基对碱性蛋白酶产生的影响，结果表明，短小芽抱杆菌的最佳发酵培养基并不适合转化子PSU一BpAP/WB60。的碱性蛋白酶的产生，而且PSU一BpAp/wB600的酶活在3种培养基更多还原

【Abstract】 Bacillus pumilus UN-31-C-42 come from strain BA(06), which is separated from life waste in ChengDu, after many times mutation. From the result of small-scale and enlarged experiments, it is indicated that the fermentation has high dehairing activity and low collagen-degradation activity. This result suggests that the protease produced by this strain might be ideal for leather industry. The protease DHAP has been purified, which can accomplish the whole dehairing process lonely.The AP gene has been cloned, which has complete coding region and partial promoter region. In order to validate whether the AP gene code the protease DHAP and the expression product of it can accomplish the whole dehairing process lonely, and construct the engineering bacteria of the AP gene , the study is performed on the expression of AP gene in B. subtilis WB600. The study includes four parts: the construction of the transform system of B. subtilis WB600, cloning of the strong promoter from bacillus, the construction of expression plasmid of AP gene and the expression of AP gene in B. subtilis WB600.In the first part, the following work was performed. The growth curves of B.pumilus UN-31-C-42 and B. subtilis WB600 were measured. The sensitivity of the two stains to the two kinds of antibiotic was determined.They were both sensitive to kanamycin. So, the kanr gene was chosen to be the marker of the transform system of them. B.pumilus UN-31-C-42 was sensitive to chloramphenicol, while B. subtilis WB600 could grow at 20 g / mL chloramphenicol. The kanr gene was cloned from the E.coli - B.subtilis shuttle vector pSUGV4, and the homology of nucleic acid between it and the one of pUB110 from Staphylococcus aureus is 100%. The competent-cell, protoplast and electroporation transformation methods were used to transform B. subtilis WB600 with the vector pSUGV4.With the competent-cell transformation methods, when the OD560 reach 1.0, the culture was transformed into MD media to cultivate, the high transformation efficiency 1.4 X 10/ g DNA was obtained. The effect of concentration of lysozyme and how long it works to the protoplast formation rate and regeneration rate were studied on B. subtilis WB600. When lysozyme concentration is 0.5mg/ml, and it works for 1 hour at 37 , the protoplast formation rate will be almost 100% and the transformation efficiency was 1.0 xio3 g DNA. Several factors affecting electroporation transformation efficiency were investigated. The parameters that resulted in high transformation efficiency were: OD560 0.8, electricity intensity 16KV/ cm, buffer SHMG , capacitance 25uF, resistance 200 , 60uL cell volume and the transformation efficiency was 2.4 x 102 gDNA.In the second part, the following study work was performed. The promoter fragments were obtained from B.pumilus UN31-C-42 and B.subtil is WB600 with the promoter-probe vector pSUPV4 in E.coli. There were 5 strong promoter fragments cloned from B.pumilus UN31-C-42, which were named Bp21 Bp38, Bp53 Bp57 and Bp70. There were 3 strong promoter fragments cloned from B.subtilis WB600, which were named Bs2, Bs4 and Bs8. These fragments were sequenced and analyzed respectively and they all contained some conserved sequences of prokaryotic gene promoters. The most efficient promoter fragment Bp53 from B.pumilus UN31-C-42 could promote the alkaline protease gene of B.pumilus expression not only in E.coli but also in B.subtilis cells. While, the most efficient Bs2 fragment from B.subtilisWB600 could promote the alkaline protease gene expression in E.coli and it also could promote the kanr gene expression in B.subtilis cells. The constitutive promoter P43 and the inducible promoter and signal peptide of the SacB gene were cloned from B.subtilis WB600, which were sequenced and analyzed. The nucleic acid of them has 100% homology to the sequence reported in Genbank. They could not promote the AP gene expression in B.subtilis WB600.In the third part, the following study work was performed. The coding region and the terminator of AP gene about 1.3kb and the Bp53 promoter fra更多还原