基因工程酶法生产谷胱甘肽
发布时间：2018-11-14 17:57
【摘要】：谷胱甘肽是一种重要的非蛋白巯基物质,在食品、化妆品和医药等领域得到了广泛的应用。本课题从经过基因工程法改造的重组菌出发,对参与体外合成GSH的谷胱甘肽双功能合成酶(GshF)和偶联ATP再生相关的多聚磷酸激酶(PPK)的表达方法进行了优化,并研究了杂蛋白表达较多的粗酶液的分离方法和分离特性。最后针对研究过程中包涵体现象较严重的多聚磷酸激酶进行了模拟结构分析,并对一类更有优势的PPK酶进行了定向进化研究,显著提高了酶的活力。具体的研究内容如下：(1)为了提高表达量,本课题首先对重组质粒的表达菌种进行优化,利用了BL21、Rosetta、OrigamB三种E.coli表达菌对比诱导表达,发现Rosetta菌的效果最好。针对PPK酶的包涵体现象,通过重新构建不同载体的重组质粒pETDuet-ppk,获得的酶用于体外合成GSH的产量提高了1.718倍。同时利用分子伴侣与目的酶共表达以促进目的酶的可溶性表达,重组质粒pETDuet-ppk同分子伴侣共表达后效果明显,获得的PPK酶用于体外合成GSH的产量提高至1.481倍,PPK酶酶活提高至1.820倍。(2)GshF酶和PPK酶的粗酶液的整体纯度较低,需要对粗酶液进行分离纯化研究。本课题实验确定了GshF酶和PPK的最适硫酸铵盐析饱和度为40%。盐析沉淀中的盐成分对酶活产生的抑制现象可以通过联合30KD聚醚砜膜的超滤处理完成有效的脱盐和浓缩。冷丙酮对GshF粗酶液可纯化倍数为1.445,收率为87.64%；冷丙酮和盐析共同处理时纯化倍数可达到1.637,收率为88.99%。(3)PPK酶表达中的包涵体现象限制了酶表达量和酶活,不仅影响了酶法体外合成GSH的整体产量,也不利于PPK酶分离纯化的研究。本课题通过结构模拟分析的方法对比了不同家族的PPK酶,研究发现Class Ⅱ PPK(PPK2)酶比实验中采用的Class Ⅰ PPK(PPK1)酶在ATP再生中更具优势。在此基础上对PPK2酶进一步定向进化,建立了高通量筛选方法及优化易错PCR突变率,使用易错PCR改造的突变株最高可提高酶活1.771倍,比活提高1.665倍。
[Abstract]:Glutathione is an important nonprotein sulfhydryl substance, which has been widely used in food, cosmetics and medicine. Based on the recombinant bacteria modified by genetic engineering, the expression methods of glutathione bifunctional synthase (GshF) and polyphosphokinase (PPK) associated with the regeneration of ATP in vitro were optimized. The separation methods and characteristics of crude enzyme with more protein expression were also studied. In the end, the polyphosphokinase (PPK), which is characterized by serious inclusion bodies, was analyzed, and a more advantageous PPK enzyme was studied by directional evolution, which significantly improved the activity of the enzyme. The specific research contents are as follows: (1) in order to improve the expression quantity, the expression strains of the recombinant plasmid were optimized in this paper. The three E.coli expressing bacteria were used to induce the expression, and the results showed that the Rosetta bacteria were the best. According to the inclusion body phenomenon of PPK enzyme, the yield of recombinant plasmid pETDuet-ppk, obtained by reconstructing different vectors for GSH synthesis in vitro was increased by 1.718 times. At the same time, the soluble expression of the target enzyme was promoted by co-expression of the molecular chaperone and the target enzyme. The effect of co-expression of the recombinant plasmid pETDuet-ppk with the molecular chaperone was obvious. The yield of the obtained PPK enzyme for GSH synthesis in vitro was increased to 1.481 times. The activity of PPK enzyme increased to 1.820 times. (2) the whole purity of the crude enzyme solution of GshF enzyme and PPK enzyme was low, so it was necessary to separate and purify the crude enzyme solution. The optimum saturation of ammonium sulfate for GshF enzyme and PPK is 40%. The inhibition of salt composition in salting-out precipitation on enzyme activity can be effectively desalted and concentrated by ultrafiltration of 30KD polyethersulfone membrane. The purified ratio of cold acetone to GshF crude enzyme solution was 1.445, and the yield was 87.64; The purification multiple of cold acetone and salting-out could reach 1.637, and the yield was 88.99. (3) the inclusion body phenomenon in the expression of PPK enzyme restricted the amount of enzyme expression and enzyme activity, which not only affected the whole yield of GSH synthesis by enzymatic method in vitro. It was also unfavorable to the study of PPK enzyme separation and purification. In this study, PPK enzymes of different families were compared by structural simulation analysis. It was found that Class 鈪，
本文编号：2331883
[Abstract]:Glutathione is an important nonprotein sulfhydryl substance, which has been widely used in food, cosmetics and medicine. Based on the recombinant bacteria modified by genetic engineering, the expression methods of glutathione bifunctional synthase (GshF) and polyphosphokinase (PPK) associated with the regeneration of ATP in vitro were optimized. The separation methods and characteristics of crude enzyme with more protein expression were also studied. In the end, the polyphosphokinase (PPK), which is characterized by serious inclusion bodies, was analyzed, and a more advantageous PPK enzyme was studied by directional evolution, which significantly improved the activity of the enzyme. The specific research contents are as follows: (1) in order to improve the expression quantity, the expression strains of the recombinant plasmid were optimized in this paper. The three E.coli expressing bacteria were used to induce the expression, and the results showed that the Rosetta bacteria were the best. According to the inclusion body phenomenon of PPK enzyme, the yield of recombinant plasmid pETDuet-ppk, obtained by reconstructing different vectors for GSH synthesis in vitro was increased by 1.718 times. At the same time, the soluble expression of the target enzyme was promoted by co-expression of the molecular chaperone and the target enzyme. The effect of co-expression of the recombinant plasmid pETDuet-ppk with the molecular chaperone was obvious. The yield of the obtained PPK enzyme for GSH synthesis in vitro was increased to 1.481 times. The activity of PPK enzyme increased to 1.820 times. (2) the whole purity of the crude enzyme solution of GshF enzyme and PPK enzyme was low, so it was necessary to separate and purify the crude enzyme solution. The optimum saturation of ammonium sulfate for GshF enzyme and PPK is 40%. The inhibition of salt composition in salting-out precipitation on enzyme activity can be effectively desalted and concentrated by ultrafiltration of 30KD polyethersulfone membrane. The purified ratio of cold acetone to GshF crude enzyme solution was 1.445, and the yield was 87.64; The purification multiple of cold acetone and salting-out could reach 1.637, and the yield was 88.99. (3) the inclusion body phenomenon in the expression of PPK enzyme restricted the amount of enzyme expression and enzyme activity, which not only affected the whole yield of GSH synthesis by enzymatic method in vitro. It was also unfavorable to the study of PPK enzyme separation and purification. In this study, PPK enzymes of different families were compared by structural simulation analysis. It was found that Class 鈪，
本文编号：2331883
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