| Compared with conventional chemical catalysis, enzyme catalysis is environment friendly, can be performed under milder and simpler process conditions, and it has higher selectivity, has been widely applied in the fields of organic matters and pharmaceuticals. Thus, enzyme catalysis is suggested to be a sustainable synthesis method for future development. One challenge for the commercial use of enzyme catalysis is the lower catalytic property, thermostability and stability in organic solvent than in water. Another problems is demanded to solve that how enzymes adapt to strong acides, base in extreme conditions.To selecte effective small molecule additives for improvements the catalytic activity of the enzymes and enhancements its stability, which was my research direction in our research group in recent years. The results of those study based on sustained research, the research orientation in the study of inorganic metal ions on the enzymes catalysis. The choice was considered to chloroperox--idase was the most versatile enzyme of heme peroxidase family (chloroperoxidase, referred to CPO) for the performance of modified target enzyme. It was repressentative of the 22 kinds of metal ions at a periodic table of elements, the main results were as follows:1. The study of 22 kinds of metal ions M+(Li+, Na+, K+, Rb+, Cs+,Ag+), M2+(Zn2+, Ni2+, Cu2+, Cd2+, Mn2+, Ca2+, Mg2+, Co2+, Hg2+, Pb2+) and M3+(Cr3+, La3+, Fe3+, Ce3+, Y3+, Al3+) on the catalytic activity of CPO showed a function of "promotion at low concentration and inhibition at high one" phenomenon. Trace metal ions (1×10-6-2×10-5mol·L) have an activation, this activation are significantly correlated with the concentration and charges of metal ions. Metal ions M3+ is the best activator in the best concentration, chloride activity of CPO catalytic MCD (2- chloro-5,5-dimethyl-1,3-cyclohexanedione) can be enhanced 17.4%-26.4%. At 50℃, incubated for 2 hours, CPO residual activity cound retain only 40% whereas 62%,75%,80% in presence of Li+,Zn2+,Cr3+. The presence of metal ions, based on UV absorption, intrinsic fluorescence and circular dichroism study of the CPO's active site microenvironment and protein secondary structure changes, in order to clarify structural basis for this activation. The results showed that, CPO's active site of the porphyrin ring increasing the degree of exposure, the substrate was conducive to close, prompting increased its activity; and enhanced a-helix structure was to enhance tolerance of the CPO for resistance high temperature and inactivation.2. Based on the activation of metal ions, and we also investigated the structure of its CPO modification. CPO's heme active center near by crystal structure prediction of existed Mn2+, the paper removed by EDTA ligand competition Mn2+, in order to direct graphite furnace atomic absorption testing confirmed the presence of Mn2+, and investigated the catalytic activity of CPO removal Mn2+ and structural changes; and then further to activate the effect of better Cr3+, Zn2+ and Ca2+ substitution Mn2+, also investigated the enzymatic properties of modified enzyme.3. An analytic method for trace chloroperoxidase was proposed based on the oxidation of fluorophore, tryptophan and tyrosine, in acid solution to enhance the conjugated effect and enhance the molecular fluorescence spectroscopy of enzyme. This method had a wide linear range (8.53×10-7-4.90×10-6 mol·L-1) and good sensitivity. The detection limit was as small as 6.83×10-7 mol·L-1. The concentration determined by this method was more Sensitive identical to that obtained from the measurement of Soret absorption of UV-vis spectrum. The relative standard deviation was less than 1.0%.Those conclusion, which is significant and valuable can direct applications the metal ions modified other enzymes to improve the catalytic performance. |
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