High-level Expression Of Recombinant Catalase And Studies On Activity Of Anti-oxidative Injuries

Catalase[hydrogen-peroxide:hydrogen-peroxide oxidoreductase(EC1.11.1.6)] is one of antioxidant enzymes and involved in many human diseases.Many studies have shown that catalase has the mitigation and treatment of acute oxidative damage. From the future of scientific research and treatment perspective,human catalase has a higher value than heterologous catalase from other origins.But the currently human catalase is mainly extracted from human blood,have not had access to be obtained in large-scale.In this study,we extracted the total RNA from human fetal liver tissue, obtained the cDNA and constructed a new pichia pastoris yeast expression to secrete the recombinant human catalase.The results showed that,we have successfully established a new recombinant yeast expression system which can secrete human catalase,the yield reached the average of 600 U / ml and the maximum could go to 1400 U / ml in a 10L fermentor.The purification of the recombinant protein involves three simple steps that yield nearly homogeneous recombinant catalase with an approximately 69%recovery and 21 purified fold.Dot-blot and western-blot reaction showed a positive result of the recombinant products when we used anti-human catalase monoclonal antibody to react with the recombinant products and supernatant of untransformed fermentation medium.This study provided a benefit explore in preparation of human catalase in large scale.Now Facing the problem of lacking of enough capability to acquire catalase in a large scale for industrial purposes,we designed a new strategy to solve it.In this study we transformed the recombinant plasmid containing Bacillus katA gene into the host strain to overexpress and secrete the recombinant catalase.The results showed that a new catalase expression system was established in B.subtilis.The purification of the recombinant protein involves three simple steps that yield nearly homogeneous recombinant catalase with an approximately 70%recovery.The purified catalase had a specific activity of 34,600 U/mg,similar to that of catalases from tissue or from other expression systems.The recombinant catalase carries higher acid and thermostability than bovine liver catalase.This study provides a new and efficient method of preparing catalase for various applications.At present the research reports about functions of catalase against acute oxidative injury are more concentrated in cells,vitro organs,and few the overall small animal experiments.The research about exogenous catalase for anti-aging in long-term has not been reported yet.In this study,we assessed the role of exogenous Catalase in the mouse model of influenza virus pneumonia and D-galactose-induced subacute-aging mice.The results showed that catalase could play an obvious role against oxidative injuries.In the influenza virus pneumonia mouse model:the treatment group was superior to the virus group in the rate from lung disease,pulmonary index,lung tissue slice,protection of life,which means exogenous catalase has great role of anti-acute oxidative damage via inhalation.In the D-galactose-induced aging mice model: intraperitoneal injection of PEG-CAT could down-regulate the level of total antioxidant capacity and MDA stimulated by oxidative stress in brain tissue,liver, heart,kidneys organizations.SOD activity of the liver could be improved,these differences were significant.The results show that catalase could become a valuable reference for potential drugs.As short half-life of catalase limits its role and application,some modifications made to enzyme are therefore very necessary.At present the most common of modification is PEG-conjugation.But administration of PEG-CAT is systemic medication,not positioning and not releasing in long term due to its half-life of dozens of hours.Studies of microsphere for delivering proteins and polypeptide vaccine are very eye-catching and different diameters of the microspheres can be achieved a variety of effects such as positioning and long-term releasing after injection.In this study,we used PLA/PEG(poly-lactic acid/polyethylene glycol) microsphere to encapsulate catalase and studied the characterizations of this microsphere.The results showed that we have succeeded in the preparation of the catalase microspheres,the rate for embedding more than 90%,observed the influence of different proportion of microspheres,outside pH,temperature and other factors on the release behavior,and made an assessment of catalase Microspheres,The experiment will be a useful exploration of delivering catalase in long-term.