Structural Stability And Molecular Modification Studies On Ascorbate Peroxidase Of Chinese Kale

Ascorbate peroxidase (APX, EC1.11.1.11), also known as vitamin C peroxidase, is animportant antioxidant enzyme in response to external oxidative stress defense and in activeoxygen metabolism in plant cell. APX is also regarded as a model for understanding therelationship between structure and function of peroxidases. On the basis of recombinant APXof Chinese kale (BaAPX) which had been obtained in our previous research, the enzymeactivity, structure, function and molecular modification of BaAPX were studied in this paper.The major results are as follows:The specific activity of APX was77.37U g-1under the optimal induced expressionconditions (30℃,10h and0.05mM IPTG) through orthogonal experiment which was1.6times compared with that through single factor optimization. The addition of ferric chloride(hemin) into purified enzyme improved BaAPX activity. Compared with control, the specificactivity of BaAPX was twice at the concentration of5mM hemin.The research on the secondary structure by circular dichroism showed that BaAPX was afull α-type enzyme with more than40%α-helix and less than5%β-fold. In addition, thespecific activity was positively related to the percentage of α-helix and the maximumpercentage of α-helix contributing to the active sites in all secondary structures was60.5%.During denaturation of BaAPX, three kinds of structural changes were proposed: the one-stepstructural change from initial state (N state) to minimum state of α-helix (R state) under lowconcentration and low temperature; the one-step structural change from N state to equilibriumstate (T state) under high concentration and low temperature; the two-step structural changesfrom N state through R state to final T state under heat treatment and low temperaturerenaturation. In inactivation rate equation of BaAPX, pre-exponential factor k0was0.1min-1and activation energy Ea was14.28KJ mol-1at20~50℃, meanwhile pre-exponential factor k0was0.0027min-1and activation energy Ea was4.65KJ mol-1at60~90℃.As stabilizers, magnesium sulfate and gelatin have better and synergistic protectiveeffect for BaAPX. The specific activities were1.7and1.9times compared with no additionunder the optimized conditions with the mass ratios of400:1or800:1between magnesiumsulfate and BaAPX or between gelatin and BaAPX respectively. Protective effects ofstabilizers on BaAPX are mainly achieved by reducing the exposure of internal groups.The acidic aspartic acid171(Asp) in amino acid sequence of BaAPX was mutated intobasic histidine (His) by PCR site-directed mutagenesis. Compared with natural BaAPX, thepercentage of α-helix and the specific activity in mutant BaAPX remained unchanged andincreased2.5%respectively.