Alpha-amylase assay and uses thereof

Abstract

This invention relates to methods and kits for measuring α-amylase activity in samples such as flour, stock, or amylase concentrate.

1 . A method for measuring α-amylase activity in a sample, comprising 
 forming a reaction mixture by contacting a sample with a detectably labeled starch substrate for a time sufficient for α-amylase in the sample to hydrolyze the starch substrate, thereby releasing soluble detectably labeled starch fragments,    separating the soluble detectably labeled starch fragments from the reaction mixture, and    determining the level of hydrolysis of the detectably labeled starch substrate as a measurement of α-amylase activity in the sample.    
   
   
       2 . The method of  claim 1 , wherein the sample is selected from the group consisting of a flour sample, a stock sample, and an amylase concentrate sample.  
   
   
       3 . The method of  claim 1 , wherein determining the level of hydrolysis of the detectably labeled starch substrate comprises quantifying the detectably labeled starch substrate.  
   
   
       4 . The method of  claim 1 , wherein determining the level of hydrolysis of the detectably labeled starch substrate comprises quantifying the soluble detectably labeled starch substrate fragments.  
   
   
       5 . The method of  claim 1 , further comprising calculating the α-amylase activity in the sample by correlating the quantity of detectably labeled starch to an α-amylase standard.  
   
   
       6 . The method of  claim 1 , further comprising calculating the α-amylase activity in the sample by correlating the quantity of soluble detectably labeled starch fragments to an α-amylase standard.  
   
   
       7 . The method of  claim 1 , wherein the detectably labeled starch substrate is potato starch.  
   
   
       8 . The method of  claim 1 , wherein the detectably labeled starch substrate comprises d-glucose residues and is labeled on about one of every 300-1300 d-glucose residues of the starch substrate.  
   
   
       9 . The method of  claim 1 , wherein the starch substrate is detectably labeled with a label compound selected from the group consisting of fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, and bioluminescent molecules.  
   
   
       10 . The method of  claim 9 , wherein the label is a fluorophore.  
   
   
       11 . The method of  claim 10 , wherein the fluorophore is selected from the group consisting of 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (EDANS), fluorescein isothiocyanate (FITC), and Marina Blue.  
   
   
       12 . The method of  claim 1 , wherein the step of separating the soluble detectably labeled starch fragments from the reaction mixture comprises filtering the reaction mixture to remove from the mixture detectably labeled starch substrate.  
   
   
       13 . The method of  claim 12 , wherein the step of filtering includes the addition of a filtration aid selected from the group consisting of resin, glass beads, beads, and celite.  
   
   
       14 . The method of  claim 1  , wherein the step of separating the soluble detectably labeled starch fragments from the reaction mixture comprises centrifuging the reaction mixture to remove from the mixture detectably labeled starch substrate.  
   
   
       15 . The method of  claim 14 , further comprising measuring an aliquot of the supernatant of the centrifuged reaction mixture.  
   
   
       16 . The method of  claim 1 , wherein the step of separating the soluble detectably labeled starch fragments from the reaction mixture comprises obtaining an aliquot of the reaction mixture and centrifuging the aliquot of the reaction mixture to remove from the aliquot detectably labeled starch substrate.  
   
   
       17 . The method of  claim 1 , wherein the step of separating the soluble detectably labeled starch fragments from the reaction mixture comprises contacting the fragments with an agent that binds to the detectably labeled starch fragments.  
   
   
       18 . The method of  claim 17 , wherein the agent is a lectin.  
   
   
       19 . The method of  claim 17 , wherein the agent is an antibody.  
   
   
       20 . The method of  claim 1 , wherein the sample is an aqueous slurry.  
   
   
       21 - 24 . (canceled)  
   
   
       25 . A method for measuring α-amylase activity in a sample, comprising 
 forming a reaction mixture by contacting a sample with a detectably labeled starch substrate attached to a surface, for a time sufficient for α-amylase in the sample to hydrolyze the starch substrate, thereby releasing soluble detectably labeled starch fragments,    separating the soluble detectably labeled starch fragments from the reaction mixture, and    determining the level of hydrolysis of the detectably labeled starch substrate as a measurement of α-amylase activity in the sample.    
   
   
       26 - 51 . (canceled)  
   
   
       52 . A kit for measuring α-amylase activity in a sample, comprising 
 a first container containing a detectably labeled starch substrate, a second container containing an α-amylase standard, and instructions for measuring the α-amylase activity in the sample.    
   
   
       53 - 73 . (canceled)  
   
   
       74 . A method of determining amylase in a sample comprising: 
 placing about 6 ml incubation buffer in a substrate tube,    warming the substrate tube to 45° C.,    adding about 200 mg of the sample to the warmed substrate tube,    incubating the sample mixture in the substrate tube 10 min at 45° C.,    adding about 4 ml stop buffer to the sample mixture in the substrate tube,    filtering the stopped sample mixture into a container,    determining the fluorescence in the filtrate, and    optionally converting the fluorescence value into a Falling Number Equivalent value.    
   
   
       75 - 79 . (canceled)  
   
   
       80 . A method of determining amylase in a sample comprising: 
 placing an about 3 g sample into a first container,    adding a sufficient amount of fungal incubation buffer to have the total weight of sample plus buffer equal of about 30 g,    mixing the solution,    extracting the solution for 5 minutes at 45° C.,    adding the about 8 ml of the extract to a substrate tube,    incubating extract in substrate tube 10 minutes at 45° C.,    adding about 2 ml stop buffer the tube,    mixing the contents of the tube,    filtering the mixture into a second container,    determining the fluorescence in the filtrate, and    optionally converting the fluorescence value into an Enzyme Units Equivalent value.    
   
   
       81 - 87 . (canceled)  
   
   
       88 . A method of determining amylase in a sample comprising: 
 placing about 200 mg of the sample into a container,    adding about 20 ml fungal incubation buffer to the sample,    mixing the sample solution,    diluting about 2 ml of the solution with 10 ml incubation buffer in a container,    optionally further diluting the diluted solution to obtain a concentration within range of about 0.1-1.0 SKB unit/ml,    placing about 8 ml of the diluted sample into a container,    incubating the about 8 ml diluted sample 10 minutes at 45° C.,    adding about 2 ml stop buffer to the 8 ml diluted sample,    filtering the mixture through a filter into a detection container,    determining the fluorescence in the filtrate, and    optionally converting the fluorescence value into an Enzyme Units Equivalent value and multiplying by the dilution factor as a measure of the original amylase concentration.    
   
   
       89 - 95 . (canceled)