【作者】 夏书琴；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 硕士

【摘要】 谷氨酰胺转胺酶(Transglutaminase,EC 2.3.2.13,简称TGase)可以催化多种蛋白质及氨基酸或谷氨酸残基之间的共价交联或酰基转移反应。由于该酶在食品、固定化酶、生物医药及纺织皮革加工等工业领域的应用,引起了人们极大的关注。本研究以提高茂源链轮丝菌(Streptoverticillium mobaraense)03-10发酵生产TGase的产量为目标,对S.mobaraense 03-10进行诱变,筛选高产突变株,并进行发酵条件优化研究。主要内容如下：(1)以S.mobaraense 03-10为出发菌株,采用大气压辉光放电的冷等离子体技术对链霉菌孢子进行诱变。根据双层平板法菌落显色及诱变处理后菌落形态差异快速筛选TGase高产突变株。突变率、正突变率分别达到42.8%和20.6%。最后复筛选育出具有较好遗传稳定性和形态稳定性的高产突变株G2-1,酶活达到2.73 U/mL,比出发菌株提高了82%。(2)对突变株S.mobaraense 03-10 G2-1种子培养基、种子培养条件及发酵培养基进行了优化。优化后的结果为：种子培养最佳碳源为25 g/L可溶性淀粉,最佳的孢子接种量106个/mL,最佳的种龄及最佳的种子接种量分别为20 h和8%。发酵培养基中最佳钙离子为2g/L无水氯化钙,最佳单一碳源为30g/L甘油,优化后的摇瓶条件下酶活达到3.71 U/mL,与优化前相比(2.71 U/mL)提高了40%。(3)考察了发酵培养基中混合碳源对发酵的影响。得出最佳的混合碳源为甘油与玉米淀粉；并比较了不同搅拌转速下不同混合碳源配比时3 L罐中发酵的情况,得出当搅拌转速为500 r/min,甘油与玉米淀粉比例为10：20时,酶活最高为3.54 U/mL。(4)在3 L发酵罐上研究了不同pH控制策略和甘油流加策略对突变菌株发酵生产TGase的影响。确定发酵过程中pH控制策略为：初始pH7.4,发酵过程中用NH3·H20控制pH≥6.5；考察了不同初始甘油浓度及不同甘油恒速流加速率下对发酵过程的影响,确定了10-20 h控制甘油流加速率为1.67g/L/h,20-30 h流加速率为0.83g/L/h,30-40 h流加速率为0.41g/L/h的分阶段甘油流加策略,与优化前结果相比较(酶活2.07 U/mL,生产强度42.71 U/(L·h)),酶活和生产强度获得了很大的提高,分别达到3.69 U/mL,76.88u/(L·h)。更多还原

【Abstract】 Transglutaminases are a family of enzymes (EC 2.3.2.13) that catalyse the formation of covalent cross-links between a free amine group and the y-carboxamide group of protein-bound or peptide-bound glutamine. It has gained interest due to its attractive potential applications in food sector, immobilization of enzymes, biomedical engineering, textiles and leather processing.This study is mainly aimed at enhancing the production of TGase by Streptoverticillium mobaraense 03-10. Firstly we mutagenized and selected the TGase producing strain and then optimized fermentation conditions. The main results are listed as following:(1) The atmospheric pressure glow discharge (APGD) plasma jet driven by a radio frequency (RF) power was used to treat the spores of S.mobaraense 03-10 for the selection of TGase producer. The mutant with high TGase production was quickly screened according to the formation of color on the double-layered plate and the different appearances of colonies. The total mutation rate was over 42.8% and the positive mutant rate was 20.6%. The obtained mutant G2-1 has good genetic and morphology stability and TGase activity reached 2.73 U/mL, which was 82% higher than that of original strain.(2) The effects of environmental conditions on fermentation by the mutant strain S. mobaraense 03-10 G2-1 were optimized in flask culture. The proper culture conditions in seed medium were as the following:the carbon source was 25 g/L soluble starch, and inoculation spores was 106per mL, and the seed age was 20 h, and the inoculum size was 8%. The optimal fermentation conditions were 2 g/L CaCl2 and 30 g/L glycerol as single carbon source. The TGase activity in flask reached 3.71 U/mL, which was improved by 40%.(3) The effects of different mixture carbon sources on fermentation were investigated. The optimal mixture carbon source were glycerol and corn starch in flask culture. Then compared the fermentation situations in different agitator speeds and different ratios of carbon sources in a 3 L stirred fermentor, the best results were that the agitator speed was 500 r/min, and the ratio was 10:20. The maximum activity was 3.54 U/mL.(4) Influence of different pH and feeding strategies on fermentation in a 3 L stirred fermentor by the screened mutant were investigated. The optimal pH control strategy in fementation processing was as the following:the initial pH7.4 and using NH3·H2O to control pH≥6.5. Based on the optimal pH control strategy, the effect of different initial glycerol concentrations and glycerol feeding rates were studied. A three-stage feeding-shift strategy was proposed:feeding rate 1.67 g/L/h during 10～20 h,0.83 g/L/h during 20～30 h,0.41 g/L/h during 30～40 h. By applying this feeding-shift strategy in fermentation, the maximal activity and productivity were significantly improved and reached 3.69 U/mL and 76.88 U/(L-h) respectively, compared with the results of before optimization(2.07 U/mL and 42.71 U/(L-h)).更多还原