【作者】 杨晓燕；

【导师】 周哲敏；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 硕士

【摘要】 谷氨酰胺转氨酶(Transglutaminase, EC 2.3.2.13,简称TGase)能催化蛋白质分子内、分子间发生酰基转移反应,从而改变蛋白质结构和功能特性,在食品、生物医药、组织工程、纺织等领域具有广泛的应用前景。链霉菌TGase是酶原(Pro-TGase)经活化而成的,但是关于链霉菌TGase的活化机理还不清楚。为了深入地研究吸水链霉菌(Streptomyces hygroscopicus)产TGase的活化机理,本论文对S. hygroscopicus发酵产生的Pro-TGase和TGase的高效分离纯化及TGase的活化过程进行了研究。主要研究结果如下：1.在培养基中分别添加不同浓度的Cu2+, Mn2+, Zn2+, Fe2+, Co2+五种金属离子,摇瓶发酵培养发现,Cu2+, Mn2+, Zn2+对S. hygroscopicus产TGase有明显的促进作用,并且确定0.05 mmol/L Cu2+是最适合添加的离子。确定了S. hygroscopicus利用优化培养基液体培养过程中,培养60 h时Pro-TGase可以完全转化成TGase。2.利用硫酸铵沉淀和阴离子交换层析以及凝胶过滤层析对S. hygroscopicus发酵上清液中的Pro-TGase和TGase进行了高效分离纯化,并得到两种活性较高而电荷量不同的TGase (TGase A和TGase B)。本研究是首次利用阴离子交换层析法对吸水链霉菌Pro-TGase和TGase分离纯化。3.通过Hiprep 16 10 phenyl FF柱和LC-MS以及CD谱对两种TGase的疏水性、天然相对分子质量以及二级结构进行分析,发现TGase A和TGase B的疏水性、相对分子质量和二级结构均存在差异。研究了两种TGase的动力学,发现两种TGase的Vmax、Km和Kcat差异也很大。4.测定了S. hygroscopicus来源的Pro-TGase的N端氨基酸序列为ASGGDG, TGase A和TGase B的N端前五个氨基酸序列均为DAADE。S.hygroscopicus来源Pro-TGase的N端氨基酸序列与已报道的S. cinnamoneus、S.fradiae、S. netropsis来源的相似性很高,但TGase的N端序列与这三种链霉菌来源的相似性却不高。本研究是首次对S.hygroscopicus来源的TGase的N端氨基酸序列进行报道。5.对纯化后的Pro-TGase与TGase A和TGase B的相互作用进行研究发现,Pro-TGase可以被自身的TGase B活化但不能被TGase A活化。热处理后的Pro-TGase对TGase A和TGase B的活性均有抑制作用,而且对TGase B的抑制作用更显著。本研究是首次对S. hygroscopicus来源的Pro-TGase可以被自身的TGase活化进行报道。6.分别对不同培养时间点的发酵液进行分离纯化,发现了TGase的活化机理：S.hygroscopicus经过24 h的培养后Pro-TGase被分泌到胞外,一部分Pro-TGase经胞外的活化蛋白酶切割成为成熟的TGase B; TGase B也具有活化自身的Pro-TGase的功能,被切去酶原区的部分空间结构发生变化,生成性质不同的TGase A。更多还原

【Abstract】 Transglutaminase (TGase; EC 2.3.2.13) is a functional enzyme, which could introduce covalent cross-links between proteins as well as amines and peptides by catalyzing acyl transfer reactions. It has great wide applications in the fields of food processing, biomedical engineering, material science, textiles. It has been reported that Streptomyces TGase is secreted as zymogen (Pro-TGase) and activated by protease, but the mechanism of TGase activation was still unclear. To further explore this mechanism, the purification and activation process of TGase from S. hygroscopicus were studied in this paper.1. S. hygroscopicus was cultured in fermentation liquid added Cu2+, Mn2+, Zn2+, Fe2+ and Co2+ of different concentration respectively. Through the analysis of TGase activity, the activity of TGase could be promote by Cu2+, Mn2+, Zn2+, and the optimal was Cu2+in the concentration of 0.05 mmol/L. After cultured 60 h, Pro-TGase could entirely transformed into mature TGase in optimized culture.2. Two active TGases (TGase A and TGase B) and Pro-TGase were effectively purified from Streptomyces hygroscopicus fermentation liquid by ammonium sulfate precipitation, Hitrap Q HP and Superdex 7510/300GL columns. This is the first report for the purification of TGase and Pro-TGase using anion chromatography.3. The two TGases exhibit different hydrophobicities, molecular weights, and second structures by analysis using Hiprep 1610 phenyl FF、LC-MS and CD spectra. They also have different Vmax, Km, and kcat values.4. The N-terminal amino acid sequence of purified two TGase were determined as DAADER and N-terminal amino acid sequence of Pro-TGase was ASGGDG. According to the amino acid sequence alignment, the N-terminal sequences of Pro-TGase from S. hygroscopicus exhibited significant sequence similarity to those from S. cinnamoneus, S. fradiae and S. netropsis, but the N-terminal sequence of TGase from Streptomyces hygroscopicus exhibited low sequence similarity to them. This is the first report of the N-terminal amino acid sequence of TGase from Streptomyces hygroscopicus.5. The results of interaction between Pro-TGase and TGase indicated that Pro-TGase could be activated by one type of mature TGase (TGase B), while the activity of both TGases was inhibited by the heat-treated Pro-TGase.6. The activation process was studied using the fermentation liquid in different time. The results indicate that Pro-TGase could be activated by some enzyme to TGase B, then TGase B activate the Pro-TGase to TGase A.更多还原