Effects Of Macromolecular Crowding On Enzymatic Hydrolysis And Activity Changes Of Protease

The paper belongs to the project of the 13 th Five-Year Project of the Ministry of Science and Technology(2018YFD0400300).Protease can hydrolyze protein and produce amino acids and polypeptides,which are widely used in food industry.In the process of protein enzymolysis,higher substrate concentration(2~7%,w/w)is usually added to improve equipment utilization,reduce energy consumption,and reduce production costs.It is found that high concentration of substrate will reduce the rate of enzymatic hydrolysis.In recent years,most scholars have only focused on methods to improve the rate of enzymatic hydrolysis.In fact,high-concentration substrates cause the "macromolecular crowding effect",and there are few reports on the changes in enzymatic hydrolysis and enzyme activity from the perspective of macromolecular crowding.In this paper,protease is used as the research object,and the effect of macromolecular crowding reagent Ficoll 70 on the activity and hydrolysis of protease from Bacillus sp.,trypsin and pepsin is explored.Endogenous fluorescence spectrum,circular dichroism and molecular dynamics simulation were used to explore the effect of macromolecular crowding on protease structure.At the same time,a sensor for the determination of macromolecules crowding on the inner surface of cell membranes was designed to make it possible to quantify the environment of macromolecules crowding on the inner surface of cell membranes.The main contents and results of this paper are as follows:1.The effects of Ficoll 70 on the hydrolysis of BSA were investigated.It was found that the degree of hydrolysis(DH)of BSA was significantly reduced after Ficoll 70 was added.The greater the amount of Ficoll 70 added,the more obvious the reduction effect.Among them,the DH of protease from Bacillus sp.decreased the most.After 3 hours of hydrolysis,it decreased from 18.2% without Ficoll 70 to 2.99%with 30%(w/w)Ficoll 70(P<0.05).Tricine-SDS-PAGE results also showed that the2.crowded environment significantly reduced the production of small molecule peptides.3.The effects of Ficoll 70 on the activity of three proteases were investigated.The results showed that Ficoll 70 significantly reduced the enzyme activity.After adding 20%(w/w)Ficoll 70,the activity of protease from Bacillus sp.decreased by49.82%,the trypsin activity decreased by 51.20% and the pepsin activity decreased by69.47%(P<0.05).At the same time,experiments on the kinetics of the enzymatic reaction were performed.Both Km and Vmax of protease from Bacillus sp.decreased,and Km and Vmax of trypsin and pepsin both increased first and then decreased.4.Explore the changes of protease structure after Ficoll 70 was added.When Ficoll 70 was added,the maximum wavelength red shift or double peak phenomenon occurred in the endogenous fluorescence spectrum of protease;The results of circular dichroism chromatography showed that the addition of Ficoll 70 did not cause significant changes in the secondary structure content;The results of molecular dynamics simulations showed that the crowded environment changed the environment of the amino acids around the fluorescence-related sites,and reduced the change in the angle and distance between the fluorescent sites and the enzyme active sites.Therefore,it is believed that the changes of protease activity and enzymatic hydrolysis in crowding are closely related to the changes of protease structure.5.Design of macromolecular crowding sensor on the inner surface of cell membrane.According to the design of macromolecular crowding sensor based on FRET principle by Boersma et al,the transmembrane region(DPP4,ACE and AQP)was introduced and fixed on the inner surface of cell membrane.After adding 200 mM sodium chloride for 10 min,YFP / CFP was significantly increased.The most significant increase in ACE-MC was 19.31%(P<0.05).It was found that the three sensors can characterize the crowding signal,and through membrane protein extraction and fluorescence microscope photo experiments,it was verified from the side that DPP4-MC in three sensors can achieve good expression on the cell membrane.