高产海藻糖菌种选育及生产工艺的研究
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摘要
海藻糖是由二个葡萄糖分子组成的非还原性双糖。由于它具有对生物大分子非特异性的保护作用，从而在食品、医药、生物化学等领域得到了广泛的应用。面包酵母(Saccharomyces cerevisiae)是生产海藻糖的重要菌种。在酵母生长后期或在不良环境的条件下可积累大量海藻糖，但由于是胞内产物，积累量有限，大大影响了产量的提高。本文利用诱变方法筛选出一株高产海藻糖的酵母菌株，并利用加热处理法使酵母海藻糖合成酶系固定化，生产胞外海藻糖，以达到提高产量的目的。研究内容主要包括海藻糖测定方法的研究、高产海藻糖菌株的选育、培养基与培养条件的优化、胞外海藻糖的合成以及海藻糖的提取与精制等。
本文对海藻糖的测定方法进行了系统的研究。提出了“纸层析-分离洗脱法”、“酶-DNS比色法”定量测定海藻糖的方法，该方法简便、成本低、准确度高。
通过对酵母菌耐性的研究表明，乙酸耐性和渗透压耐性与海藻糖含量之间有一定的正相关性，具有较高海藻糖含量的菌株可以在高乙酸和高渗透压的生长环境中更好地生存。综合耐性实验和海藻糖积累实验的结果，确定了海藻糖含量高，渗透压和乙酸耐性最强的Y7菌株为本研究出发菌株。实验对出发菌株Y7进行了EMS诱变处理，通过初筛、复筛，从中选育出一株能高产海藻糖同时具有较低海藻糖分解酶活性的菌株L61。与原菌株Y7相比较，对数期比生长速率提高22.7％，酸性海藻糖分解酶活性下降19.2％，中性海藻糖分解酶活性下降26.7％，胞内海藻糖含量提高了57.2％。
培养条件对海藻糖合成影响较大，本文对影响海藻糖合成的各种因素进行了优化。应用正交设计、Plackett-Burman实验设计和响应曲面分析法确定了最佳的培养基配方，并对反应条件进行了优化研究。在优化条件下，菌株L61摇瓶反应3h，产海藻糖0.82g/L，比初始条件下海藻糖积累量(0.56 g/L)提高了46.4％。
以摇瓶培养最优条件为基础，进行了摇瓶补料实验。在初糖浓度20g/L的条件下，反应过程中补加底物葡萄糖有利于海藻糖的合成。在糖总量不变的情况下，补料培养的海藻糖合成量比非补料培养增加了50.4％。
本文首次研究了利用酵母海藻糖合成酶系合成胞外海藻糖的可行性。研究表明，50℃高温处理不仅有利于海藻糖合成酶系活性的发挥，还可使细胞壁受到一定的破坏，从而使海藻糖自由透过胞壁进入反应液，打破了胞壁对海藻糖
接二一激二二一人二．～一～…回．一二～… ～－＋。一、上＿一二二人二、－．一。二～～．一二二回一二一二一．二～二：一二…．二一比二j王立二二二竺
摘要
合成的空间限制。研究还发现，选用5％接种量，3％初糖浓度，反应过程中底
物浓度 1％左右，pH6刀，50’C条件下震荡反应 6h，海藻糖合成量可达不94g／L。
以摇瓶补料为基础，深入进行了SL罐胞外海藻糖流加合成实验。确定了流
加合成胞外海藻糖的最适条件，在底物浓度1％，pH6．0，温度50C条件下，发
酵 6h，可使海藻糖的合成总量达 14．sg／L，比摇瓶反应的海藻糖合成量增加了
62．2％。同时研究还发现，虽然在反应过程中不断产生海藻糖，但在反应结束时，
菌体胞内的海藻糖几乎为零，说明菌体的胞壁对海藻糖的出人已无任何障碍，
菌体只是成为海藻糖合成酶的载体，通过不断地补加底物使海藻糖得以不断合
成。
提取是反应过程的最后一道工序，本文对酵母胞内外的海藻糖提取方法进
行了初步研究。通过超滤、离于交换等步骤提取出高纯度结晶体（纯度为98％人
经红外吸收光谱和TLC鉴定，证明结晶体为海藻糖。
利用酵母海藻糖合成酶系合成胞外海藻糖是海藻糖生产方法的一个创新，
它结合了酶法和酵母提取法的优点，是利用酵母大量生产海藻糖的又一新的尝
试。
Trehalose is a nonreducing disaccharide with two glucose residues. Because trehalose exhibit protective action against damage of high molecular substances , it achieve widespread application in a variety of fields including foods, pharmaceutics and biochemistry etc . Baker's yeast (Saccharomyces cerevisiae) is a major strain of trehalose production. Trehalose is accumulated at the end of the reproductive stages and under extreme environmental conditions. Because the trehalose is intracellular product, its productivity is limited. This paper examines the breeding of the mutant with high-yielding trehalose and the production of extracellular trehalose via the immobilized trehalose synthase. The main research contents include the methods for trehalose determination, the breeding of trehalose high-producing strain, the optimization of medium and culture conditions, the production of extracellular trehalose and the purification of the intra- and extracellular trehalose.
The methods for trehalose determination were studied in this paper. The methods of paper chromatography-mottle elution cnromometry and enzyme-DNS colorimetric were defined. These methods were simple, cheap and accuate .
The results showed that the positive correlation was obtained between trehalose concentration and acetic acid or osmotic tolerant. Through the test, the strain of Y7, which had the highest content of trehalose and strong tolerance to sodium chloride and acetic acid, was defined as a parental strain. In order to enhance the trehalose production, Y7 was used for mutation with EMS. Through primary screening and compound screening, strain L61 with high-yielding trehalose and deep neutral trehalase activity was screened. Comparing with the Y7, the specific growth rate of mutant increased by 22.7%, the activities of neutral and acid trehalase droped by 19.2%and 6.7%, respectively. Under the non-optimized condition, the production of trehalose increased by 57.2% compared with that of parental strain Y7.
The factors affecting the trehalose production were studied in this paper. The Placket-Burman design method and Respond Surface analysis method were used to optimize the medium and the reaction conditions were optimized too. Under the optimized condition strain L61 could produce 0.82g/L trehalose after reaction for 3 hours by flask- shaking batch culture. The production of trehalose increased by 46.4% compared with that of the initial condition (0.56g/L). An investigation has been carried out of fed batch culture according to the optimum condition of batch culture. The intermittent feeding glucose was helpful to the trehalose production in the condition of 20g/L initial glucose concentration. The trehalose production of the fed batch culture
increased by 50.4% compared with that of flask- shaking batch culture.
In the yeast, the heat shock of 50 癈 can be helpful to the increase of the activity of trehalose synthase, on the other hand it can destroy yeast's cell wall. The intracellular trehalose produced could excreted to the culture medium. Keeping the pH at 6.0, 3% of the initial glucose concentration , 5% of seed volume, 1% of glucose concentration, heat shock at 50 癈 for 6 hours resulted in 8.94g/L trehalose.
Based on the flask-shaking fed-batch culture, the test of the fed-batch reaction which synthesized the extracellular trehalose was performed with strain L61 in 5-liter bioreactor. The optimized factors were defined. Keeping the pH of the culture broth at 6.0,the initial glucose concentration of l%,the strain could produce 14.5g/L trehalose after reaction at 50 for 6 hours, the production of trehalose increased by 62.2% than that of flask-shaking culture. Meanwhile, the results showed that althougt the trehalose was produced successively , the intracellular trehalose concentration was 0 at the end of the reaction. Trehalose was excreted to culture medium completely. Yeast's cell was only as a carrier of the trehalose synthase. Trehalose could be synthesized successively with the feeding of glucose.
Extraction
本文对海藻糖的测定方法进行了系统的研究。提出了“纸层析-分离洗脱法”、“酶-DNS比色法”定量测定海藻糖的方法，该方法简便、成本低、准确度高。
通过对酵母菌耐性的研究表明，乙酸耐性和渗透压耐性与海藻糖含量之间有一定的正相关性，具有较高海藻糖含量的菌株可以在高乙酸和高渗透压的生长环境中更好地生存。综合耐性实验和海藻糖积累实验的结果，确定了海藻糖含量高，渗透压和乙酸耐性最强的Y7菌株为本研究出发菌株。实验对出发菌株Y7进行了EMS诱变处理，通过初筛、复筛，从中选育出一株能高产海藻糖同时具有较低海藻糖分解酶活性的菌株L61。与原菌株Y7相比较，对数期比生长速率提高22.7％，酸性海藻糖分解酶活性下降19.2％，中性海藻糖分解酶活性下降26.7％，胞内海藻糖含量提高了57.2％。
培养条件对海藻糖合成影响较大，本文对影响海藻糖合成的各种因素进行了优化。应用正交设计、Plackett-Burman实验设计和响应曲面分析法确定了最佳的培养基配方，并对反应条件进行了优化研究。在优化条件下，菌株L61摇瓶反应3h，产海藻糖0.82g/L，比初始条件下海藻糖积累量(0.56 g/L)提高了46.4％。
以摇瓶培养最优条件为基础，进行了摇瓶补料实验。在初糖浓度20g/L的条件下，反应过程中补加底物葡萄糖有利于海藻糖的合成。在糖总量不变的情况下，补料培养的海藻糖合成量比非补料培养增加了50.4％。
本文首次研究了利用酵母海藻糖合成酶系合成胞外海藻糖的可行性。研究表明，50℃高温处理不仅有利于海藻糖合成酶系活性的发挥，还可使细胞壁受到一定的破坏，从而使海藻糖自由透过胞壁进入反应液，打破了胞壁对海藻糖
接二一激二二一人二．～一～…回．一二～… ～－＋。一、上＿一二二人二、－．一。二～～．一二二回一二一二一．二～二：一二…．二一比二j王立二二二竺
摘要
合成的空间限制。研究还发现，选用5％接种量，3％初糖浓度，反应过程中底
物浓度 1％左右，pH6刀，50’C条件下震荡反应 6h，海藻糖合成量可达不94g／L。
以摇瓶补料为基础，深入进行了SL罐胞外海藻糖流加合成实验。确定了流
加合成胞外海藻糖的最适条件，在底物浓度1％，pH6．0，温度50C条件下，发
酵 6h，可使海藻糖的合成总量达 14．sg／L，比摇瓶反应的海藻糖合成量增加了
62．2％。同时研究还发现，虽然在反应过程中不断产生海藻糖，但在反应结束时，
菌体胞内的海藻糖几乎为零，说明菌体的胞壁对海藻糖的出人已无任何障碍，
菌体只是成为海藻糖合成酶的载体，通过不断地补加底物使海藻糖得以不断合
成。
提取是反应过程的最后一道工序，本文对酵母胞内外的海藻糖提取方法进
行了初步研究。通过超滤、离于交换等步骤提取出高纯度结晶体（纯度为98％人
经红外吸收光谱和TLC鉴定，证明结晶体为海藻糖。
利用酵母海藻糖合成酶系合成胞外海藻糖是海藻糖生产方法的一个创新，
它结合了酶法和酵母提取法的优点，是利用酵母大量生产海藻糖的又一新的尝
试。
Trehalose is a nonreducing disaccharide with two glucose residues. Because trehalose exhibit protective action against damage of high molecular substances , it achieve widespread application in a variety of fields including foods, pharmaceutics and biochemistry etc . Baker's yeast (Saccharomyces cerevisiae) is a major strain of trehalose production. Trehalose is accumulated at the end of the reproductive stages and under extreme environmental conditions. Because the trehalose is intracellular product, its productivity is limited. This paper examines the breeding of the mutant with high-yielding trehalose and the production of extracellular trehalose via the immobilized trehalose synthase. The main research contents include the methods for trehalose determination, the breeding of trehalose high-producing strain, the optimization of medium and culture conditions, the production of extracellular trehalose and the purification of the intra- and extracellular trehalose.
The methods for trehalose determination were studied in this paper. The methods of paper chromatography-mottle elution cnromometry and enzyme-DNS colorimetric were defined. These methods were simple, cheap and accuate .
The results showed that the positive correlation was obtained between trehalose concentration and acetic acid or osmotic tolerant. Through the test, the strain of Y7, which had the highest content of trehalose and strong tolerance to sodium chloride and acetic acid, was defined as a parental strain. In order to enhance the trehalose production, Y7 was used for mutation with EMS. Through primary screening and compound screening, strain L61 with high-yielding trehalose and deep neutral trehalase activity was screened. Comparing with the Y7, the specific growth rate of mutant increased by 22.7%, the activities of neutral and acid trehalase droped by 19.2%and 6.7%, respectively. Under the non-optimized condition, the production of trehalose increased by 57.2% compared with that of parental strain Y7.
The factors affecting the trehalose production were studied in this paper. The Placket-Burman design method and Respond Surface analysis method were used to optimize the medium and the reaction conditions were optimized too. Under the optimized condition strain L61 could produce 0.82g/L trehalose after reaction for 3 hours by flask- shaking batch culture. The production of trehalose increased by 46.4% compared with that of the initial condition (0.56g/L). An investigation has been carried out of fed batch culture according to the optimum condition of batch culture. The intermittent feeding glucose was helpful to the trehalose production in the condition of 20g/L initial glucose concentration. The trehalose production of the fed batch culture
increased by 50.4% compared with that of flask- shaking batch culture.
In the yeast, the heat shock of 50 癈 can be helpful to the increase of the activity of trehalose synthase, on the other hand it can destroy yeast's cell wall. The intracellular trehalose produced could excreted to the culture medium. Keeping the pH at 6.0, 3% of the initial glucose concentration , 5% of seed volume, 1% of glucose concentration, heat shock at 50 癈 for 6 hours resulted in 8.94g/L trehalose.
Based on the flask-shaking fed-batch culture, the test of the fed-batch reaction which synthesized the extracellular trehalose was performed with strain L61 in 5-liter bioreactor. The optimized factors were defined. Keeping the pH of the culture broth at 6.0,the initial glucose concentration of l%,the strain could produce 14.5g/L trehalose after reaction at 50 for 6 hours, the production of trehalose increased by 62.2% than that of flask-shaking culture. Meanwhile, the results showed that althougt the trehalose was produced successively , the intracellular trehalose concentration was 0 at the end of the reaction. Trehalose was excreted to culture medium completely. Yeast's cell was only as a carrier of the trehalose synthase. Trehalose could be synthesized successively with the feeding of glucose.
Extraction
引文
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