| The enzyme target molecular screening techniques have been extensively used in China and abroad.Existing enzymes target methods in screening mainly inspect the filter object on enzyme inhibition,therefore requiring high enzyme activity.Patient's diseased tissue was the common way to get the high activity enzymes,and only can be provided in small quantities.Genetic engineering can clone enzymes,but often low activity.It is difficult to be used in the existing drug screening method which depend on the active of inhibition.According to the current researches,no enzyme activity screening techniques requires to be found.The laboratory has been established a method which can be used for screening of natural active substances on frontal affinity chromatography screening platform.The experiments in the paper are based on the platform,to screen natural medicines which used enzyme as target.Continue to rely on the frontal affinity chromatography technology,to the level of structural integrity,but low dynamic activity enzyme as a screening target.Through the research on the influence of enzyme activity to screening results,we obtained screening application in different conditions of enzyme activities,and verify the screening platform in the enzyme target level structure under conditions of intact but low activity,which can still achieve effective results.Also the method is applied to the Glucoamylase inhibitor detection,this method is more precise and more sensitive than the traditional Glucoamylase inhibitor screening method.The main contributions were as follows:Experiment methods:1.In order to study if the enzyme activity can influence on screening results.Trypsin is heated,and obtained level structure does not change but different activities of enzymes.The first object is the substrate specificities of trypsin N-A-benzoyl-DL-arginyl-4-Nitro aniline hydrochloride(BAPNA).The enzyme activity of 2933.33 U/mL and 33.33 U/mL trypsin were used as target enzymes,gel-sol method column is used.And compared the test results can obtain the effect on the result of enzyme activity.2.Applied frontal affinity chromatography to trypsin inhibitor screen-ing,Matrine and Oxymatrine has been selected as objects accordi-ng to our laboratory's pre-verification.On the basis,similarly,heating to get the enzyme at different activities:2933.33 U/mL,2053.33 U/mL,1353.33 U/mL,1030 U/mL,373.33 U/mL,66.67 U/mL,33.33 U/mL,32.33 U/mL enzyme.Made these enzymes which at different activites as target,matrine and oxymatrine as objects,can get results of screening on the effect of enzyme activities.3.Base on the above experiments,we established frontal affinity chromatographic screening method used Glucoamylase as target enzymes,and myricetin as checking object.The application on Glucoamylase is made under different conditions of enzyme activities,enzyme purity and PEG dosage.Experimental results:1.The Kd(0.525?M)of BAPNA and trypsin which activity is 2933.33 U/mL was significantly smaller than Kd(1.977 ?M)when the trypsin activity is 33.33 U/mL.That means when trypsin activity reduced,the combination of BAPNA and trypsin become lower.The results validate enzyme specificity of trypsin and substrate.2.Experimental results show that,when enzyme activity changes,the Kd of matrine oxymatrine and enzyme have small but nor obvious changes.At the same time combination between small molecular substances and enzymes is likely to be the unit combination,and more correlated to the pace structure of enzyme.3.The results show that,enzyme activity significantly reduced,but Kd of myricetin and Glucoamylase has no obvious change;when Glucoamylase increase the purity,the Kd of myricetin and Glucoamylase increased obviously;when the molecular weight of the PEG in a certain range(400-1000),the smaller molecular weight,the better outflow curve;when molecular weight same,the PEG concentration is higher,the sample elution curve breakthrough size is larger.In the range of 150 mg/mL-250 mg/mL,all can obtain effective results. |
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