Study On The Effect Of Ionic Strength On Enzyme Activity

The paper belongs to the project of the 13th Five-Year Project of the Ministry of Science and Technology?2018YFD0400300?.The electrostatic interaction of ionic strength has a very broad influence on the function of biological macromolecules inside and outside the cell.Enzymes in biological macromolecules play an important role in the nutritional regulation of the human body and food processing?marinated,dairy products?.However,the ionic strength makes a great effect on the electrostatic interaction of the microenvironment for enzyme,and then affects the exertion of the enzyme action.This study presents the study on the protease from Bacillus sp.,porcine pancreatic?-amylase,and?-galactosidase in vitro to investigate the effect of ionic strength on its activity and mechanism.At the same time,the internal ionic strength changes with time were observed by the means of changing the ionic strength in the culture medium of HEK293 cells,and the effect of intracellular ionic strength changes on the activity of superoxide dismutase?SOD?in HEK293 cells is also studied in details.The main research contents and results of this article are as follows:?1?Studies on the effect of ionic strength?NaCl?on the activity of protease from Bacillus sp.,and show that ionic strength sodium chloride?NaCl?has an inhibitory effect on the protease.As the ionic strength increases,the inhibitory effect becomes stronger and both the enzymatic kinetics parameters K_m and V_max are decreased.The principle was further explored by SDS-polyacrylamide gel electrophoresis,scanning electron microscopy,Fourier transform infrared spectroscopy,fluorescence spectroscopy,circular dichroism,and molecular simulation.The results show that the protease interacts with the ions in the environment resulting in electrostatic interaction and rough surface,the functional groups including-C=O and-C-N,and the hydrogen bond of?-sheet of protease is severely broken,and the ratio of parallel and anti-parallel structures is changed,the larger the anti-parallel ratio,the less parallel,causing a decrease in protease activity.?2?Studies on the effect of ionic strength?NaCl?on?-amylase activity of pig pancreas,and found that the sodium chloride?NaCl?had an inhibitory effect on?-amylase with the increase of ionic strength,and the enzymatic kinetic parameters of K_m and V_max are reduced.To further explore its principles through SDS-polyacrylamide gel electrophoresis,scanning electron microscopy,Fourier transform infrared spectroscopy,fluorescence spectroscopy,circular dichroism,and molecular simulation.The results show that?-amylase interacts with ions in the environment make the surface rough,the functional groups including-C=O and-COO^-,and in the secondary structure,the helical structure increases first and then decreases,and the folding decreases first and then increases,and affects the catalytic group of the active center of the enzyme,which leads to a decrease in?-amylase activity.?3?Studies on the effect of ionic strength?ZnSO₄?on?-galactosidase activity,and found that with the increase of ionic strength,zinc sulfate?ZnSO₄?had an inhibitory effect on?-galactosidase,and the enzymatic kinetic parameters of K_m and V_max are reduced.Further explored its principles through SDS-polyacrylamide gel electrophoresis,scanning electron microscopy,Fourier transform infrared spectroscopy,fluorescence spectroscopy,circular dichroism,and molecular simulation.The results show that?-galactosidase generates electrostatic interaction with Zn²⁺in the microenvironment,and changes the functional groups-CH₂ and-COO^-.At the same time,the catalytic center of the enzyme is also affected by Zn²⁺,which leads to a decrease in?-galactosidase activity.?4?By culturing HEK293 cells with a medium containing 200 mM NaCl,it was found that the intracellular ionic strength continued to increase with the increase of the culture time,and the declining trend was greater In the first 30 min,and gradually stabilized after that.Observing the morphology of cells cultured under 200 mM NaCl medium under a microscope,it was found that some cells appeared in an unclear outline state,and the SOD enzyme activity was stable within 30 minutes,but the SOD enzyme activity was inhibited after 30 minutes.In summary,the ionic strength of the microenvironment for enzyme has high influence on the electrostatic interaction between the enzyme and the ions,as well as the activity of the enzyme.To investigate the effect and mechanism of ionic strength on enzyme activity in vitro,makes a good the foundation for exploring the mechanism of enzyme activity change in vivo,and plays a significant role in functional foods for nutritional regulation?such as blood glucose,digestion and absorption,and lactose intolerance?.