Genetic Engineered And Enzymatic Method For Glutathione Synthesis

Glutathione is an very important non-protein sulfhydryl compounds which has been widly applied in food/cosmetics/medicine. This research starts from recombinant bacteria which is transformed by genetic engineering and optimize the expression of Bifunctional glutathione synthesase (GshF) that takes part in GSH synthesis in vitro and Polyphosphate kinase (PPK) related with coupled ATP regeneration. And then we research on the separation methods and characteristics of crude enzyme which obtains lots of hybrid protein. At last, the research carries out simulative structural analysis to Polyphosphate kinase which presents more serious Inclusions phenomenon found in the our research and takes process in directed evolution of the PPK enzyme which owns more advantages, then significantly increased enzyme activity. The specific study is as followed. 1. For the purpose of improve expression quantity, this research first optimize the expression strain of recombinant plasmid, using three kinds of E.coli expression strain as BL21,Rosetta and OrigamB to compare expression, finding out that the effect of Rosetta is the best. Aiming at the Inclusion body phenomenon of PPK enzyme, through reconstructing recombinant plasmid pETDuet-ppk as a new carrier, improve the yield of GSH synthesis in vitro by 1.718 times. At the same time, using chaperone co-expression with the target enzyme to reduce inclusion body. The effect is more obvious after recombinant plasmid pETDuet-ppk and chaperone co-express. The production of GSH which enzyme PPK taking part in synthesis improves by 1.418 times, and the activity of enzyme PPK improves by 1.820 times.2. The lower overall purity of the crude GshF and crude PPK lead us to study on separation and purification of the crude enzyme solution. The experiment determined that the optimal salting saturation of enzyme GshF and PPK is 40 percent. The salt composition in the precipitation after salting out will inhibit the enzyme activity. We can joint ultrafiltration process using 30 kD poly ether sulfone membrane to complete effective desalination and concentration. Cold acetone can purify crude enzyme GshF to purified ratio of 1.445 with the yield of 87.64 percent,especially for jointing with salting-out, the purified ratio can be up to 1.637 with the yield of 88.99 percent.3. The expression efficiency and enzyme activity of the target enzyme were affected by the inclusion generated in the PPK expression.It not only limits the overall yield in vitro enzymatic synthesis of GSH, but also restricts the separation and purification of enzyme PPK. This paper compares the different families of enzyme PPK by the method of structure simulation analysis. The study found that Class II PPK (PPK2) are better than Class I PPK (PPK1) used in the experimental in hydrophilicity. It is difficult to form inclusion bodies in the expression of enzyme PPK2, and it more likely to transform polyphosphate into ATP. Meanwhile, we remould the enzyme PPK2 by directed evolution. Finally,we established a high-throughput screening method and Optimize the error-prone PCR mutation rate. The best mutant after transformation by error-prone PCR will increase enzyme activity up to 1.771 times with the pecific Activity up to 1.665 times.