The Chemical Study For The Enzyme Activity And Stability Of Nattokinase

Nattokinase isolates from traditional food natto.It has strong fibrinolytic and thrombolytic activity and is expected to be a potential medicine for preventing and treating cardiovascular disease.Improving the activity and stability of nattokinase to enlarge their clinical and commercial application range has attached more and more attention of researchers.Metal ions play critical and fundamental roles as cofactors with catalytic functions or structural support elements in biology.Thus,adding some metal ions to enzymes has become one of the principal means to change the activity of nattokinase.It is evident that various metal ions influenced nattokinase activity in different degrees with a concentration range from 1 to 10 mM.Although Metal ions are essential for all living organisms in proper concentration,they presenting in excessive amount could have toxic effect.High concentration of metal ions can affect the structure of protein,lead to protein denaturation or aggregation,and even inactivation.It inspires us to undertake systematic studies on the effect of metal ions on structure and function of nattokinase at the molecular level.The paper relies on UV-Visible spectra,resonance light scattering spectra,fluorescence spectra,chemical unfolding experiments and the measurement of enzyme activity to investigate the interaction of nattokinase with metal ions and the impact of metal ions on the activity and stability of nattokinase at the molecular level.In this study,spectroscopy was used to study the binding of metal ions with nattokinase and the aggregation of nattokinase.The results showed that nattokinase was capable of binding with Cu²⁺,Co²⁺,Mn²⁺ and Tb³⁺ in stoichiometric ratio,respectively.The binding constants were also obtained by double-logarithm regression equation.Moreover,the ionic competition experiment indicated that there is a binding site of competition in NKbetween Tb³⁺ and Ca²⁺.The high concentration of Cu²⁺ and Co²⁺ resulted in the aggregation of the nattokinase.While Tb³⁺,Ca²⁺,Na+ remarkably inhibited the aggregation of nattokinase.Different concentration of metal ions were different on changing nattokinase activity.For low concentration of metal ions,the activity of nattokinase determined by tetra-peptide substrate method at proper p H and temperature was not influenced when bound with Tb³⁺ and Ca²⁺,and the inhibitory effects could be found in the catalysis activity of nattokinase due to the presence of Cu²⁺,Co²⁺ and Mn²⁺.For high concentration of metal ions,Tb³⁺ and Ca²⁺ improved the activity of nattokinase,however,Cu²⁺,Co²⁺,Mn²⁺ still inhibited activity of NK.Further,it was also found that there was a tight connection between activity and concentration of NK.To better compare the stability of NK in the absence and presence of metal ions,nattokinase samples were unfolded through continuous concentrations urea and guanidinium hydrochloride,respectively.Chemical denaturation of the protein sample were found to be a typical two-state?N?U?process.Based on the structure element model,the thermodynamic parameters for unfolding of protein were obtained using a two-state mechanism.The data showed that metal ions could not change the average structural element free energy <?G0element?H2O?> of NK by the measurement of enzyme activity,but they could improve the stability of the global protein by the fluorescence spectral measurement.That is to say,metal ions could improve the stability of nattokinase,but had no effect on the active site for enzyme.The innovation lying in the work is that a more intensive study is carried out to investigate the chemical basis for the effect of the metal ions on the enzyme activity and stability,compared with the traditional researches.We also find that the addition of the some metal ions can significantly reduce the aggregation of nattokinase,which is important for enlarging the practical value of enzyme.In addition,the stability of nattokinase was measured by thechemical unfolding experiment detected by enzyme activity and spectral measurement with the model of structural element.