| As the most efficient biocatalyst. enzymes are widely distributed in animals, plants and microorganisms. However, most natural enzymes are mesophilic enzymes and cold-active enzymes, which mean poor biological stability, are often unbearable in severe external conditions, such as high temperature, exposure to organic reagents, etc., and these conditions in most industrial processes are widely and necessarily. Therefore, for a large-scale application of natural enzymes in industrial production, the corresponding protect devices are always needed, and that will not only surge the costs, but also handicap the accumulation of positive product under low temperature. So the poor biological stability of natural enzymes has severely limited the further applications in industrial productions.For these reasons, in this study, the lipase 1 from Candida Rugosa (CRL1) was chosen as the enzyme for enhancing thermo stability, due to its wide applicability and the poor thermo stability which has hampered its further application in industrial production, but also offered the space for the improvement to its stability.First, the gene lip1 was artificially synthesized expressed in Pichia pastoris. The physicochemical and catalytic properties of the recombinant lipase were also detected, and the results indicated that the modified procedure for producing the lipase CRL1 was successful.The B-factor and rational design method were used to predict the mutant sites and LEU 302 and GLU 126 were chose as the key amino acids to enhance the stability of CRL1.In the next step, the two sites will be mutated using saturation mutagenesis. respectively. The applications of native superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are restricted by their instability and limited availability. not least because it is extremely difficult to prepare using genetic engineering techniques. However, in the second part of this thesis, a mimic of antioxidant enzymes has been produced.From examination of the steady state and pre-steady state kinetic behavior of natural GPX it was found that, in contrast to accepted theories, the affinity of the enzyme for H2O2 rather than reduced glutathione (GSH) most significantly affects its kinetic behavior.Consequently, an enzyme mimic was produced with a similar affinity for the substrate H2O2. A salicylaldehyde Schiff base containing a dimanganese centre was selected as a precursor, because it has high H2O2-binding affinity for such a relatively small molecule and similar catalytic activity to that of SOD and CAT. Selenium was also incorporated into the catalytic center to provide activity similar to that of GPX. and thus trifunctional enzymatic activity.The KmH2O2 of the mimic (7.32×10-2 mM) was found quite close to that of natural enzyme (1.0×10-2 mM). indicating that the affinity of the mimic to H2O2 was successfully increased to approach natural GPX. The steady state kinetic performance of the enzyme mimic showed that the ratio between kcat/KmGSH and kcat/KmH2O2 was quite similar to that of native GPX. indicating that the Mn(III)2(L-Se-SO3Na) had the same selectivity for both substrates GSH and H2O2 as native GPX, which put it among the best existing GPX mimics.Moreover, the new mimic was confirmed to strongly inhibit lipid peroxidation and mitochondrial swelling, probably due to the synergism between the three antioxidant enzymatic activities.All the results indicating that the strategy of artificial synthesis a mimic of antioxidant enzymes is successful.
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