Determination Of KLK Enzyme Activity By High Performance Liquid Chromatography And Screening Of Foodborne KLK Promoters

Hypertension has become the first chronic disease in China and one of the important factors endangering health.Both kallikrein(KLK)and angiotensin converting enzyme(ACE)play a very important role in blood pressure regulation,antagonizing each other and regulating the balance of blood pressure in the body.Currently,the research on ACE inhibitors has been very mature,but there is relatively little research on KLK promoters,so this article will conduct in-depth research on them.Clinical medication for hypertension is accompanied by many side effects,so follow the "drug food homology" principle to explore the effect of food extracts on KLK activity,so as to determine its effect on lowering blood pressure.In this paper,KLK enzyme was extracted from pig pancreas,and purified to obtain high purity pancreatin powder;To establish a method for determining the activity of KLK enzyme by high performance liquid chromatography,which can accurately determine the activity of KLK enzyme;50 food extracts were selected and subjected to three extraction methods to obtain food extracts.After the initial screening of the effects of food extracts on KLK enzyme activity,the best food extracts were obtained through secondary screening.Then,chromatography and preparative liquid were used to separate and purify them,and their components were identified through mass spectrometry analysis.Finally,molecular docking simulation was performed,The relationship between the stability of enzyme peptide complexes and the binding mode of enzyme peptide and the promotion effect of KLK activity was investigated.The specific contents are as follows:The KLK enzyme was extracted from pig pancreas,activated by calcium chloride,and then used isopropanol as an extracting agent,chitosan as a precipitating agent.After defatting with acetone,a crude enzyme powder was prepared,which was preserved by vacuum freeze drying.Using ammonium sulfate with 20% and 70% saturation for salting out,and then undergoing ultrafiltration to remove salt,using D354 macroporous weakly basic anion exchange resin for purification,and using SDS-PAGE to identify its purity,it was obtained that its molecular weight was about 30 k D,specific activity was 61.22 U/mg,and purification multiple was 25.78.To explore the method for determining the activity of KLK enzyme,a UV spectrophotometer was used for kinetic determination of KLK enzyme activity,but the determination process was unstable and there were many interference factors.Therefore,a method for determining the activity of KLK enzyme by high performance liquid chromatography was established,and the specificity of buffer,standard BA,and substrate BAEE was investigated.Then,precision,stability,repeatability,and recovery tests were conducted.The RSDs were calculated to be 0.42%,0.54%,0.85%,and 0.94%,respectively,with a recovery rate of 100.89%,It can be seen that using HPLC to determine KLK enzyme activity has a good effect.A total of 50 foodborne materials from three categories of fruits,vegetables,and aquatic products were selected to obtain a food extract through hydrolysis,ethanol extraction,and pepsin enzymatic hydrolysis.The effect of the extract on the promotion of KLK enzyme activity was measured.From the perspective of food types,the effect of vegetables is relatively good.From the perspective of extraction methods,water extract and enzymatic hydrolysate have better effects than alcohol extract.Among them,red extract,razor clam,ginger,blueberry,and shrimp have better hydrolysis effects,while garlic,balsam pear,spinach,flatfish,dragon head,pumpkin,cucumber fish,small yellow croaker,red extract,ginger,tomato,mango,apple,and potato have better enzymatic hydrolysis effects.After secondary screening and in vitro simulated digestion experiments,the results showed that the enzymatic hydrolysate of spinach in vitro digestion had the best promotion effect on KLK enzyme activity,with a promotion rate of 54.75%.The spinach in vitro simulated digestive enzyme hydrolysate with the best KLK activity promotion effect was isolated and purified.After Sephadex G-15 gel filtration and elution,the preparative liquid phase separation,the component with the strongest promotion effect was obtained.The purity of the component was identified using the analytical solution,and its structure was identified by LC-MS.The polypeptide sequence obtained was His Asn Ser Pro Gly Tyr Tyr Asp Gly Arg.Finally,molecular docking simulation was performed to obtain a molecular docking simulation diagram showing good binding of amino acid sequences to KLK.