Study On The Interaction Between Calcium Ions And Alkaline Protease And Its Mechanism Of Action

Alkaline protease is a protease active in the neutral to basic pH range.Alkaline proteases catalyze hydrolysis reactions that break down protein molecules into peptides and amino acids.Alkaline proteases are widely used in detergents,foodstuffs,pharmaceuticals and leather industries so that alkaline proteases become the most studied type of protease.The performance of alkaline proteases is influenced by many factors,such as pH,ionic strength,temperature,mechanical handling and so on.Alkaline proteases with excellent properties can further optimize the process and are therefore always in demand.Due to the huge industry demand,researchers are constantly exploring the different aspects of alkaline protease properties.For industrial applications,alkaline protease must also be active and stable under less favorable conditions(extreme temperatures and pH,presence of inhibitors and oxidants)and therefore protease needs process adaptability and long-term stability.As the alkaline protease used in the traditional process is unstable in quality and low in enzyme activity,the structure of the alkaline protease becomes loose immediately after pH and temperature change extremely,introduction of heavy metal ions and urea.Enzymatic activity reduce or even lose,so that the application of alkaline protease has been limited.In view of this,the activator of alkaline protease activity becomes an important factor to consider in the practical application of protease preparations.These cations can activate the activity of alkaline protease to some extent.Therefore,in this study,the alkaline protease PB92 of known structure was selected as the research object,and there were two calcium binding sites in the structure.Calcium chloride and calcium formate were selected to study the effect of calcium ions on alkaline protease.The effect of calcium chloride and calcium formate on the activity of alkaline protease PB92 was studied in this paper.The mechanism of the interaction between calcium ions and the alkaline was investigated by dynamic light scattering experiments and fluorescence spectra.First of all,the enzyme activity of alkaline protease PB92 was measured by adding different concentrations of calcium chloride and calcium formate under the optimum reaction conditions of alkaline protease.The results showed that calcium chloride and calcium formate could promote protease activity in the range of 1 mmol/L to 5 mmol/L,and calcium chloride promoted the activity of alkaline protease PB92 more obviously than calcium formate.Secondly,the changes of the size and protease solution potential of alkaline protease PB92 were determined after adding different concentrations of calcium ions by Malvin laser particle size analyzer.The change of particle size after adding different concentration of calcium ion shows that the addition of calcium ions reduced the average particle size of the alkaline protease.the hydrophobic structure gradually transferred to the internal structure of the protease which was conducive to improve the activity of protease.The potential change of protease solution after adding different concentration of calcium ion shows that the electrostatic interaction of calcium ions with the surface groups of alkaline protease can mask the excess negative charge on the surface of the alkaline protease,resulting in a gradual decrease in the zeta potential of the enzyme solution,eliminating the adverse effects of charged groups on alkaline protease.It is beneficial to maintain the activity of enzyme.Thirdly,the fluorescence intensity changes of calcium ion and alkaline protease PB92 were studied.It was found that calcium ion had a certain quenching effect on alkaline protease PB92.According to the Stern-Volmer formula,the results showed that calcium ions form a stable complex with alkaline protease.The quenching mechanism of calcium ions to alkaline protease was a single static quenching,with quenching rate constant being2.82×10~100 L/(mol·s),binding constant being 6.03x10~5 L/mol and binding site number being 1.90.Through the analysis of thermodynamic parameters calculated,we saw that calcium ions and alkaline protease form stable complexes through electrostatic force,and the binding process can proceed spontaneously.