【摘要】：谷胱甘肽是一種重要的非蛋白巰基物質(zhì),在食品、化妝品和醫(yī)藥等領(lǐng)域得到了廣泛的應(yīng)用。本課題從經(jīng)過基因工程法改造的重組菌出發(fā),對參與體外合成GSH的谷胱甘肽雙功能合成酶(GshF)和偶聯(lián)ATP再生相關(guān)的多聚磷酸激酶(PPK)的表達(dá)方法進(jìn)行了優(yōu)化,并研究了雜蛋白表達(dá)較多的粗酶液的分離方法和分離特性。最后針對研究過程中包涵體現(xiàn)象較嚴(yán)重的多聚磷酸激酶進(jìn)行了模擬結(jié)構(gòu)分析,并對一類更有優(yōu)勢的PPK酶進(jìn)行了定向進(jìn)化研究,顯著提高了酶的活力。具體的研究內(nèi)容如下：(1)為了提高表達(dá)量,本課題首先對重組質(zhì)粒的表達(dá)菌種進(jìn)行優(yōu)化,利用了BL21、Rosetta、OrigamB三種E.coli表達(dá)菌對比誘導(dǎo)表達(dá),發(fā)現(xiàn)Rosetta菌的效果最好。針對PPK酶的包涵體現(xiàn)象,通過重新構(gòu)建不同載體的重組質(zhì)粒pETDuet-ppk,獲得的酶用于體外合成GSH的產(chǎn)量提高了1.718倍。同時(shí)利用分子伴侶與目的酶共表達(dá)以促進(jìn)目的酶的可溶性表達(dá),重組質(zhì)粒pETDuet-ppk同分子伴侶共表達(dá)后效果明顯,獲得的PPK酶用于體外合成GSH的產(chǎn)量提高至1.481倍,PPK酶酶活提高至1.820倍。(2)GshF酶和PPK酶的粗酶液的整體純度較低,需要對粗酶液進(jìn)行分離純化研究。本課題實(shí)驗(yàn)確定了GshF酶和PPK的最適硫酸銨鹽析飽和度為40%。鹽析沉淀中的鹽成分對酶活產(chǎn)生的抑制現(xiàn)象可以通過聯(lián)合30KD聚醚砜膜的超濾處理完成有效的脫鹽和濃縮。冷丙酮對GshF粗酶液可純化倍數(shù)為1.445,收率為87.64%；冷丙酮和鹽析共同處理時(shí)純化倍數(shù)可達(dá)到1.637,收率為88.99%。(3)PPK酶表達(dá)中的包涵體現(xiàn)象限制了酶表達(dá)量和酶活,不僅影響了酶法體外合成GSH的整體產(chǎn)量,也不利于PPK酶分離純化的研究。本課題通過結(jié)構(gòu)模擬分析的方法對比了不同家族的PPK酶,研究發(fā)現(xiàn)Class Ⅱ PPK(PPK2)酶比實(shí)驗(yàn)中采用的Class Ⅰ PPK(PPK1)酶在ATP再生中更具優(yōu)勢。在此基礎(chǔ)上對PPK2酶進(jìn)一步定向進(jìn)化,建立了高通量篩選方法及優(yōu)化易錯(cuò)PCR突變率,使用易錯(cuò)PCR改造的突變株最高可提高酶活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，

本文編號：2331883