【作者】 吉国栋；

【导师】 高红亮；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 谷氨酰胺转胺酶能够催化体外多种蛋白质之间、蛋白质和氨基酸之间的交联反应，能够实现蛋白质的改性而提高蛋白质的功能性质和营养价值，因而在食品工业中具有广泛的应用。最初商用谷氨酰胺转胺酶主要从动物组织中提取，由于来源稀少，分离纯化工艺复杂，导致其价格很高，不可能应用于食品工业。而微生物发酵法生产谷氨酰胺转胺酶（microbial transglutaminase，简称MTG）的历史较短，还存在生产技术不完善、发酵水平不高等问题。因而要实现MTG的廉价工业化生产，还需要做大量的研究工作。 本文以实验室保藏的茂原链轮丝菌（Streptoverticillium mobaraense）为研究菌株，主要从以下几个方面研究：通过摇瓶发酵优化培养基，得出适宜工业化生产的培养基配方。然后在该培养基配方的基础上进行摇瓶分批补料发酵试验，确定最佳补料方案。根据摇瓶实验结果探讨10L发酵罐的分批补料发酵策略，为工业化生产奠定基础。根据本实验室的发酵体系，建立了一个MTG分批发酵的动力学模型，为进一步的实验提供理论指导。 通过以上几方面的研究，得到如下结果： 1．适宜工业化生产的培养基配方：可溶性淀粉：0.5％，葡萄糖：2.0％，酵母膏：0.5％，豆饼粉水解物：2.0％，硫酸铵：0.3％，硫酸镁：0.2％，磷酸氢二钾：0.2％，磷酸镁：1.0％，pH 7.4。 2．进行摇瓶分批补料发酵时，补料的最佳碳源是葡萄糖，葡萄糖补料最佳时间为发酵24h，最佳浓度为1.0％。1.0％的葡萄糖分两等份在12h和24h补加时能最大限度的提高MTG产量，其酶活5.56 u·ml^-1比对照4.6u·ml^-1高出20.87％，也高于24h一次性投加1.0％的葡萄糖的酶活5.12u·ml^-1。氮源补料时，不论是有机氮源还是无机氮源都没能起到提高酶活的效果。 3．控制发酵环境的pH值，从8h开始以不同速率进行流加补料，结果表明，低的葡萄糖流加速率(0.01 L·h^-1和0.02 L·h^-1)相对于高的流加速率(0.03L·h^-1和0.04 L·h^-1)对微生物细胞的生长、MTG的合成以及底物的利用均有利。其中，0.01 L·h^-1的葡萄糖流加速率的菌体干重和酶活分别达到23.5 g·L^-1和5.28 u·ml^-1，与控制pH值的分批发酵过程相比，菌体干重提高了8.05％，酶活提高了30.05％。 4．对MTG分批发酵建立了包括三个动力学方程的数学模型 茂原链轮丝菌的生长动力学方程更多还原

【Abstract】 The transglutaminase can catalyze acyl transfer reactions in vitro introducing covalent cross-links between proteins as well as peptides and various primary amines. This reaction can change the properties of protein, increasing function characteristics and nutrition of protein. So the transglutaminase plays more and more important role in the food technology. Commercial transglutaminase has been mainly from animal tissues in the beginning stage. The scarce source and complicated isolation and purification procedures result in an extremely high price of the enzyme. Therefore it is not possible to apply such expensive tissue-type transglutaminase into food processing on an industrial scale. Microbial transglutaminase （MTG） has been produced from microorganisms. MTG is only studied for a few years, and there are some problems in the large scale production MTG, so lots of work must be done for the MTG use in a commercial way.The main studies were taken as follows:The Streptoverticillium mobaraense fermentation medium was optimized by flask batch culture. The fed-batch fermentation method was studied by flask culture based on the optimal medium. Then the fed-batch fermentation method was studied in 10L fermenter, which founded the base for industrial production. The MTG batch fermentation model was developed base on the data of 10L batch fermentation.The main results were as follows:1. The industrial optimal meidum: Starch:0.5%, Glucose: 2.0%, Yeast stract: 0.5%, soy hydrolysis : 2.0%, （NH₄）₂SO₄ 0.3%, MgSO₂·7H₂O : 0.2%, K₂HPO₄·3H₂O: 0.2%,Mg₃（PO₄）₂:1.0%,pH7.42. In flask fed-batch fermentation, the best carbon source was glucose, and the best feed time and feed concentration was 24h and 1.0%, respectively. That glucose divided into twice and feed at 12h and 24h can get the highest MTG production, which MTG activity (5.56 u·ml^-1) was 20.87% higher than the control (4.6u·ml^-1) and also was higher than 1.0% glucose once feed at 24h (5.12 u·ml^-1). It was no obvious effect to feed nitrogen source, with organic nitrogen source or inorganic nitrogen source.3. The fed-batch fermentation was feed from 8h with pH controlled. The results showed that relative slow feeding rate (0.01 L·h^-1and 0.02 L·h^-1) promoted the cell更多还原