【作者】 冮洁；

【导师】 杜连祥；

【作者基本信息】 天津科技大学 ， 发酵工程， 2005， 博士

【摘要】 论文首先以基因工程菌株里氏木霉(Trichoderma reesei)为研究对象,对其生物合成组织型纤溶酶原激活剂(tissue-type plasminogen activator,t-PA)的发酵条件和发酵动力学进行了研究,并对里氏木霉重组t-PA的分离纯化及其性质进行了探索;然后进行了t-PA突变体的研究,获得了t-PA突变体r-PA基因,并在甲醇毕赤酵母(Pichia methanolica)中进行了表达。 采用稀释平板分离法对基因工程菌株Trichoderma reesei进行了分离纯化,对平板上生长良好的单菌落进行摇瓶液态发酵,以t-PA的酶活力为指标,挑选高产菌株,选得的Trichoderma reesei(8)号菌株t-PA产量最高,且遗传稳定性良好。 对Trichoderma reesei(8)生物合成组织型纤溶酶原激活剂(t-PA)的代谢调节机制进行了研究。在基础发酵条件下,L-山梨糖、D-蔗糖、可溶性纤维素、CMC、麦芽糖、乳糖和棉子糖等单糖及多糖对t-PA生物合成都有诱导作用,其中L-山梨糖的诱导效果最好,加量以1.0%为宜。葡萄糖及其中间代谢产物对t-PA的生物合成产生分解代谢物阻遏作用。纤维二糖在低浓度时可以促进t-PA的生物合成而在高浓度时对t-PA生物合成起反馈阻遏作用。 应用单因素和正交设计试验法,确定了种子培养基的最佳组成和最佳培养条件。应用正交设计试验法和响应面分析试验法优化出摇瓶分批发酵培养基的最佳组成。通过研究不同发酵条件对t-PA生物合成的影响,获得了最佳摇瓶分批发酵培养条件。优化条件与初始条件的摇瓶分批发酵比较试验结果表明,Trichoderma reesei(8)菌株在优化条件下的t-PA产量(3275.26U/mL)比初始条件下(1.14U/mL)显著提高。 对5L发酵罐上的分批发酵过程进行了分析。选择了适宜发酵条件并以5L发酵罐分批发酵试验数据为依据,采用MATLAB工具软件,利用全局收敛的修正的高斯-牛顿法算法,以误差平方和最小为目标,获得发酵动力学方程待估参数,建立了发酵动力学模型。通过拟合分析证明得到的数学模型误差较小,能较好的反映t-PA的分批发酵过程。 对里氏木霉重组t-PA的分离纯化方法进行了研究,建立了里氏木霉重组t-PA的分离提纯工艺。通过超滤、硫酸铵盐析、Butyl Sepharose 4 F F疏水层析、Sephadex G-25凝胶过滤脱盐、Q-Sepharose F F离子交换层析分离等分离步骤,里氏木霉重组t-PA的比活力由347.78U/mg提高到10619.58U/mg,提纯倍数30.54,活力回收率2.63%。通过SDS-PAGE电泳对各步纯化后样品进行分析,经上述各步将里氏木霉重组t-PA分离为电泳纯。 通过SDS-PAGE纤维蛋白自显影证明了Trichoderma reesei(8)的发酵液中更多还原

【Abstract】 The dissertation focuses on the fermentation conditions, the fermentation kinetics, the purification and the enzyme characteristics of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei.The clone of r-PA cDNA and expression in Pichia methanolica was also investigated.Recombinant Trichoderma reesei strain was screened through dilution-plate method. With t-PA activity as target ,the high-producing Trichoderma reesei (8) with highly genetic stability was selected by shaking flask liquid cultivation of the strains from single colony on plate.The metabolic control mechanism of tissue-type plasminogen activator(t-PA) produced by recombinant Trichoderma reesei was studied.Under the minimal fermentation conditions,the saccharides including sucrose,carboxymethyl cellulose, raffinose,L-sorbose ,lactose ,dissolubility cellulose and maltose could induce the t-PA production. L-sorbose is the best inducer when its concentration is 1.0%. Glucose and its metabolites could produce catabolite repression . Cellobiose could promote the biosynthesis of t-PA when its concentration is low and could produce feedback repression when its concentration is high.The seed medium composition and culture conditions were optimized by single factor experiment and orthogonal experiment. The shaking flask batch fermentation medium composition and conditions were also determined through orthogonal experiment and response surface methodology.The maximum t-PA yield was 3275.26 U/mL,and was improved by thousand times as great as 1.14 U/mL in original fermentation conditions.Based on the optimal conditions obtained in shaking flask, the batch fermentation was performed in 5-liter fermentor. The batch fermentation kinetics of t-PA produced by recombinant Trichoderma reesei were studied based on the experimental data from the fermentation in 5-liter fermentor. The kinetic models of fermentation were established by MATLAB software.Purification process of recombinant t-PA from Trichoderma reesei (8) was explored and established.The recombinant t-PA was purified using ultrafiltration, salting-out, Butyl-Sepharose 4FF hydrophobic interaction chromatography, Sephadex G-25 gel filtration chromatography and Q-Sepharose FF ion exchange chromatography.The purified t-PA was homogeneous examined by SDS-PAGE electrophoresis.The recombinant t-PA was analysed by SDS-PAGE fibrin autography technique .The results showed that there were two kinds of hydrolysis fibrin products in the broth of Trichoderma reesei(8)with 6.6×10~4 and 3.3×10~4 molecular weight respectively.The fibrinolytic characteristic of the recombinant t-PA was also analysed by negative and positive plates,which revealed the t-PA only activated plasminogen to plasmin.there wasnot hydrolysis fibrin product in the broth of host. The preservationtime (4°C) and the freezing-thawing times have much influence on the enzyme activity.The longer preservation time and the more freezing-thawing times ,the more enzyme activity lost. The optimum temperature of the crude and pure recombinant t-PA were 37°C and 45 °C respectively. The optimum pH of the recombinant t-PA was 9.4. The recombinant t-PA was stable in the pH range of 6.4~~8.4 at 37 "C.The isoelectric point of the recombinant t-PA was 3.82 estimated by isoelectrofocusing electrophoresis.The preparation methods of recombinant Trichoderma reesei chromosome DNA were studied.Three kinds of methods of freezing-grinding-CTAB, freezing-grinding-SDS and chloride benzyl-SDS were compared and DNA were detected by agarose gel electrophoresis.The result showed that freezing-grinding -CTAB was a better method.The preparation conditions of the method were optimized.The chromosome DNA prepared by this method meet the requirements of PCR and other molecular biological manipulation.According to the published sequence of r-PA gene, a pair of primers which can amplify the r-PA region was designed. The r-PA gene was amplified by PCR with recombinant Trichoderma reesei chromosome DNA as template, then the r-PA gene was inserted into pMD18-T vector. The recombinant plasmid was used to transform the competent E.coli DH-5a cells. The transformed cells were spread on LB agar plates with ampicillin,X-gal and IPTG to isolation of recombinant strains according to blue-white reaction. A positive clone harboring r-PA gene was identified by restriction enzyme analysis and PCR technique. Collecting recombinant plasmid pMD18-r-PA and sequencing the recombinant plasmid,the sequencing result showed that the cloned 1.1kb fragment of r-PA sequence homology was 99.91% with the r-PA gene published , the amino acid sequence was same as that published.The secreted expression plasmid pMETaA-r-PA of Pichia methanolica was constructed and digested with Pac I and transformed into Pichia methanolica PMAD16 by electroporation.The method of high efficient electrotransformation for pMETotA-r-PA to Pichia methanolica was established.The positive transformants which integrated r-PA gene in their genomes were obtained by screening on MD plates and identified by PCR technique.The Mut phenotypes of these transformants were Muts.Under shake-flask culture and induced using methanol as a carbon source, the extracellular r-PA reached the largest activity of 1675.62U/mL at 72h. The expression products were analyzed by SDS-PAGE.The results indicated that the r-PA from Pichia methanolica PMAD16 was secreted into broth and not glycosylated with 3.96 X 104 molecular weight.更多还原