| Nattokinase (NK, formerly designated subtilisin NAT) was first screened from a traditional Japanese soybean-fermented food named natto by Sumi et al in 1987. In Japan, the vegetable cheese natto is a typical and popular soybean fermented food. Its history could extend back more than 2000 years for the special taste. Traditionally, it was also used as a folk medicine for heart and vascular diseases, to relieve fatigue and as an anti-beriberi agent. NK is a subtilisin-like serine protease and is produced by food-grade microorganisms Bacillus subtilis var. natto from soybean-fermented food natto. NK has strong fibrinolytic activity and it exhibits a 4-fold greater thrombus-dissolving activity than plasmin. NK not only directly cleaves cross-linked fibrin, but also activates the production of t-PA (tissue plasminogen activator), resulting in the transformation of inactive plasminogen to active plasmin. Furthermore, NK enhances its fibrinolysis through cleavage and inactivation of PAI-1 (inhibitor of t-PA). Studies on the thrombus degradation of NK indicate that NK is a potential natural oral agent for the treatment of dissolving thrombus. Compared with conventional clot-dissolving drugs, NK has several advantages including safety, oral administration, low-cost, confirmed efficacy and prolonged effects in the gastrointestinal tract. All these observations make it has the potential to be developed as functional food additives and drugs to prevent or cure cardiovascular diseases. Therefore, using protein engineering technology to enhance the activity and stability of NK should be useful for broading the utility of NK in medical and commercial applications. During this study, we first used molecular biological technology and biochemical methods to study the expression of NK mature peptide gene [aprN (MP)] and NK gene (aprN) in Escherichia Coli (E. coli) BL21. We further investigated the function of signal peptide and propeptide for the expression and maturing of NK in E. coli BL21. Then, catalytic residue Ser221 was replaced by Cys and Ala respectively by utilizing the method of overlap extension PCR. The results showed that the process of maturing precursor to active enzyme not only needed the function of signal peptide and propeptide but also needed the activity of enzyme itself to autolyze the propeptide which is a potent competitive inhibitor of the enzyme.The 131L mutant was constructed by site-directed mutagenesis based on the molecular dynamics (MD) simulation. Ni-NTA column and DEAE sepharose fast flow column were used to purify target proteins and active enzymes were successfully purified to homogeneity. The purified enzymes were analyzed with respect to kinetic parameters and fibrinolytic activity. The kinetic parameters of enzymes were investigated by spectroscopy assay and isothermal titration calorimetry (ITC), and fibrinolytic activities were determined by fibrin plate method. Spectroscopic studies together with ITC showed consistent kinetic characterizations and indicated that substituting Leu for lle at position 31 resulted in about 2-fold increase in catalytic efficiency(Kcat/KM) compared with the wild-type NK. Furthermore, The MD simulation in present work showed that the 131L mutation made Asp32 move closer to His64, and thus strengthened the negative charge on the imidazole ring of His64 This strong negative charge could increase the alkalinity of NE2 (His64). At the same time, the strong attraction of NE2 (His64) for the proton of hydroxyl group of Ser221 caused strong nucleophilic attack. Thus, the rate of conversion of substrate into product was increased and the catalytic efficiency improved.Four mutants (T220S, M222A, T220S/I31L and M222A/I31L) were constructed by site-directed mutagenesis. NK variants were purified by techniques of column chromatography using Ni-NTA column and DEAE sepharose fast flow column and active enzymes were successfully purified to homogeneity. Target proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis. The purified enzymes were analyzed with respect to kinetic parameters, fibrinolytic activity and oxidative stability, respectively. The kinetic parameters of enzymes were measured with the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA (suc-AAPF-pNA) and fibrinolytic activities were determined by fibrin plate method. Fluorescence spectroscopy measurement and circular dichroism (CD) spectra were employed to probe conformational changes induced by mutation. The results showed that the two single mutants (T220S and M222A) were found to have a greatly increased oxidative stability compared with the wild-type NK. However, the enzymic activities of both single mutants were seen to be lowered more or less compared with the wild-type NK. The two double mutants (T220S/I31L and M222A/I31L) were found to have a greatly increased enzymic activities compared with single mutants (T220S and M222A). At the same time, the oxidative stability of M222A/I31L mutant was greatly enhanced relative to the wild-type NK and is similar to single mutant M222A. On the other hand, the oxidative stability of T220S/I31L mutant was fould to decrese compared with single mutant T220S and is similar to the wild-type NK in 1.0M H2O2. Furthermore, fluorescence spectroscopy measurement indicated that the microenvironments around the fluorophore functional groups of enzymes did not change significantly induced by mutation and CD spectra also indicated that the secondary structure of proteins did not change significantly for mutation. MD simulations on the wild-type NK and T220S mutant suggested that a hydrogen bond was formed between Ser220 and Asn155, and the spatial structure of Met222 was changed compared with the wild-type NK.In order to study the effects of disulfide bond on the thermostability of NK, two cysteines were introduced to form a disulfide bond in the NK molecule and three double mutants (G61C/S98C, T22C/S87C and S24C/S87C) were constructed by site-directed mutagenesis. Target proteins and the disulfide bond were detected by SDS-PAGE and western blotting analysis. The thermostability of enzymes was measured by their rates of inactivation at elevated temperatures. The results showed that disulfide bond was not formed in the neither of three double mutants and the thermostability of mutants was also not improved compared with the wild-type NK. On the other hand, the thermostability performed in the presence of 1mM Ca2+ was much stronger than in the presence of 1mM ethylenediaminetetraacetic acid (EDTA) at the same temperature conditions. But when the temperature reached 62℃, the enzymes rapidly denatured and inactivated even in the presence of Ca2+... |
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