【作者】 谈丹；

【导师】 王金华；

【作者基本信息】 湖北工业大学 ， 发酵工程， 2008， 硕士

【摘要】 谷氨酰胺转胺酶（EC2．3．2．13，蛋白质-谷氨酸-γ谷氨酰胺转胺酶，Transglutaminase，缩写为TGase）可催化蛋白质分子间或分子内共价键的形成，从而改变各种蛋白质的功能性质，在食品、化妆品、医药等领域具有广阔的应用前景。传统上TGase主要从动物组织提取，但基于经济上的考虑，必须去寻求更便利及经济的来源，微生物发酵法原料廉价，生产成本低，是进行大规模工业化生产最有效的方法。本文主要研究了维生素对Streptoverticillium SG 215发酵生产MTG的影响，并优化了其添加量以期提高发酵单位酶活力；并在实验室优化工艺的基础上建立了其分批发酵的动力学方程；最后探索研究了微生物谷氨酰胺转胺酶对大鼠创伤的促愈合作用。顺序采用Plackett-Burrman设计、最速上升法以及中心组合方法研究了维生素B1等9种维生素对Streptoverticillium SG 215生产谷氨酰胺转胺酶的影响。结果表明泛酸钙和对氨基苯甲酸对MTG的发酵具有显著性的促进作用。中心组合优化得到发酵培养基中最佳的维生素组合是V_B1 20mg/L，V_B2 0．5mg/L，烟酸6mg/L，泛酸钙0．028mg/L，烟酸吡多醇0．50mg/L，生物素20mg/L，对氨基苯甲酸0．044mg/L，叶酸0．4mg/L，吡哆醛盐酸盐0．2mg/L，其发酵末MTG活力达2．42U/ml，较未添加维生素之前（1．58U/ml）提高了41．77％。对谷氨酰胺转胺酶生产菌Streptoverticillium SG 215分批发酵动力学模型进行了研究。建立了比较合理的谷氨酰胺转胺酶分批发酵动力学模型：细胞生长动力学模型为:（?）;MTG合成动力学模型为：（?）;葡萄糖消耗动力学模型为:（?）。模型的计算结果和试验测定值吻合较好，基本能反映发酵过程中相关指标的变化规律，可用于发酵产量的预测。最后研究了微生物谷氨酰胺转胺酶对大鼠创伤的促愈合作用。结果实验组创面愈合时间平均为18．1天，较对照组平均缩短了2-3天（P<0．05）；创伤愈合率显著提高（P<0．05或P<0．01）；伤腔容积明显缩小（P<0．05）；实验组肉芽干湿重较对照组显著增加（P<0．05），肉芽中蛋白质、氨基已糖和己糖醛酸含量增加显著（P<0．05）。结果显示谷氨酰胺转胺酶具有促进大鼠皮肤创伤愈合的作用。更多还原

【Abstract】 Transglutaminase (TGase, protein-glutamineγ-glutamyltransferase, EC. 2.3.2.13) is an enzyme that catalyses the acyl transfer reaction by introducing covalent cross-links between proteins, peptides and various primary amines. The reaction catalyzed by transglutaminase can be used to modify the function of proteins, thus transglutaminase has been widely used in food, cosmetic, pharmacy and other industries. Traditionally, most sources of TGase come from animal organs, but in view of economic value, but by the economics, it is necessary to look for more convenient and economical method for production. Fermentation has the advantage of low cost, which makes it a most promising method in large-scale production of transglutaminase.The influence of vitamin for Streptoverticillium SG 215 was systematically studied with central composite design. Firstly, the prime factors affecting MTG activity were selected by Plackett-Burrman; secondly, the prime factors were optimized by steepest ascent design and Central Composite Design. The results showed that Calcium Pantothenate and Para-amino benzoic acid had great influence on the growth of Streptoverticillium SG 215 and MTG activity, and the optimum fermentation medium were Vitamin B1 20 mg/L,Vitamin B2 0.5 mg/L, Niacin 6 mg/L, Calcium pantothenate 0.028 mg/L, Vitamin B3 0.5 mg/L, Pyridoxal hydrochloride 0.2 mg/L, Folic Acid 0.4 mg/L, Biotin 20 mg/L, Para-amino benzoic acid 0.044 mg/L. The maximum activity of MTG under optimum fermentation medium was 2.42 U/ml which was 41.77% higher than that of the original medium (1.58 U/ml).The fermentation kinetics of glutathione with SG 215 was studied. The kinetic models were obtained. The cell growth model was showed in following equation:(?),the MTG biosynthesis model was showed in followingequation:(?);the glucoseConsumption mode was showed in following equation:(?).The kineticmodel provided a reasonable description for biomass, product and substrate variation along with the fermentation process.Effect of Microbial Transglutaminase from SG215 on cutaneous wound healing in rats was evaluated using excision wound model. The closure time and volume of the skin wounds in the experimental groups significantly decreased when compared to that in the control group (P<0.05).Wound contraction, dry(or wet) weight of granulation tissues and content of protein, Hydroxyproline, Hexuranic, Hexosamines in the wound tissues in the experimental groups were higher than that in the control group (P<0.05).These results clearly substantiate the beneficial effects of microbial transglutaminase in wound healing.更多还原