【作者】 许晓娟；

【导师】 堵国成；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 硕士

【摘要】 微生物谷氨酰胺转胺酶(Microbial Transglutaminase,简称MTG,EC 2.3.2.13)能催化多种蛋白质分子间、分子内的酰基转移反应,改善各种蛋白质的功能性质,在食品、化妆品、制药等工业领域具有广阔的应用前景。本研究以提高吸水链霉菌(Streptomyces hygroscopicus)WSH03-13发酵产谷氨酰胺转胺酶的产量为目标,分别研究了吸水链霉菌的高密度培养和溶氧浓度对发酵过程的影响并通过不同的诱变手段来筛选高酶活的菌株。主要内容如下:1.对吸水链霉菌的高密度培养进行了研究。通过考察不同初始葡萄糖浓度下的分批发酵,确定了最佳初始葡萄糖浓度为10 g/L。通过比较了不同的流加速率,确定在8-16 h采用较高的的比生长速率(0.15 h-1),后期降低比生长速率(0.10 h-1)这种较优的葡萄糖流加速率组合可以获得较高的菌体量。2.考察了添加氮源对Streptomyces hygroscopicus发酵产酶的影响。在菌体生长到最大值后添加氮源,促进产酶。实验结果表明,添加50 g/L的豆饼粉酶活最高可达到5.79 U/mL。3.在3 L罐中考察了不同的溶氧浓度对谷氨酰胺转胺酶分批发酵的影响,确定了在0-20 h保持30%的溶氧浓度,通气量1.25vvm;20 h后保持10%的溶氧浓度,通气量1.25vvm的条件来进行发酵,最终酶活可达到5.88 U/mL,实现了谷氨酰胺转胺酶的高产量、高生产强度的统一。4.采用N+注入、紫外照射等常规诱变手段,并筛选抗2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)突变株。由此分离纯化获得的高产菌株DG-1具有抗葡萄糖代谢阻遏的能力。最高酶活可达到8.19 U/mL,比出发菌株提高了96%。更多还原

【Abstract】 Microbial transglutaminase (MTG; protein-glutamineγ-glutamyltransferase, EC 2.3.2.13) is a recently developed enzyme and is capable of catalyzing acyl transfer reactions, which introduces covalent cross-links between proteins, peptides and various primary amines. So it has potential application in food processing, cosmetic, pharmacy and other industries.This study is mainly aimed at enhancing the production of microbial transglutaminase (MTG) by Streptomyces hygroscopicus. The relatively high cell density culture was invesgated. The effect of DO concentration and the effect of mutagenesis of Streptomyces hygroscopicus were researched, respectively. The following researches have been done:1. The relatively high cell density culture of Streptomyces hygroscopicus was investigated, the optimized initial concentration of glucose was 10 g/L.The effects of exponential feeding of glucose and the addition of nitrogen source on MTG production were investigated. The results showed that keeping a high specific growth rate (0.15 h-1) in 8-16 h and a relative low specific growth rate (0.10 h-1) during later time could obtain high-cell density.2. Effect of nitrogen sources addition on MTG fermentation by Streptomyces hygroscopicus was investigated. Nitrogen sources were added until the end of cells growing. The results showed that MTG activity reached 5.79 U/mL by adding 50 g/L soybean.3. The effect of dissolved oxygen (DO) concentration on the batch production of MTG in a 3 L stirred fermentor by Streptomyces hygroscopicus was studied. It was found that keep the DO at 30% during 0-20 h, and 10% during later time, the air flow rate was controlled at 1.25 vvm all the time in the fermentation process, the final MTG activity reached 5.88 U/mL achieving the unification of high MTG yield and high productivity.4. Streptomyces hygroscopicu CCTCC M203062 was mutagenized by UV and N~+-Implantation, and screened by 2-deoxy-D-glucose. DG-1, which is resistant to catabolite repression, was screened. The highest activity reached 8.19 U/mL, increased by 96% over the parent strain.更多还原