Methods for detecting and measuring polysaccharide-hydrolyzing enzymes

Abstract

Methods are disclosed for detecting and measuring polysaccharide-hydrolyzing enzyme activity or concentration by partial hydrolysis using a pre-determined, yet short, incubation time and a pre-determined temperature. The resulting reaction mixture has unique chemical (i.e., reaction products) and physical (i.e., viscosity) properties that can be used to detect or measure the polysaccharide-hydrolyzing enzyme activity or concentration.

1 . A method of measuring a polysaccharide-hydrolyzing enzyme concentration, the method comprising the steps of:
 a) milling a plant part wherein the plant part contains an endogenous polysaccharide substrate;   b) suspending the milled plant part in liquid to make a solution wherein the solution comprises a polysaccharide-hydrolyzing enzyme that hydrolyses said endogenous polysaccharide substrate;   c) heating the solution to a temperature above a gelatinization temperature of the polysaccharide substrate and wherein the polysaccharide-hydrolyzing enzyme is active;   d) measuring viscosity data of the solution for at least one time point wherein the time point is at least 30 seconds;   e) comparing the viscosity data of d) with a calibration curve wherein the calibration curve is generated in the same manner using at least two calibration samples of known polysaccharide-hydrolyzing enzyme concentration;   f) measuring said polysaccharide-hydrolyzing enzyme concentration.   
     
     
         2 . The method of  claim 1 , wherein said plant part is a transgenic plant part comprising a heterologous polysaccharide-hydrolyzing enzyme. 
     
     
         3 . The method of  claim 1 , wherein b) comprises 1 or more additional polysaccharide-hydrolyzing enzymes 
     
     
         4 . The method of  claim 1 , wherein said polysaccharide-hydrolyzing enzyme is selected from the group consisting of alpha-amylase, cellulase, glucoamylase, beta-amylase, endoglucanase, pullulinase, xylanase and combinations thereof. 
     
     
         5 . The method of  claim 1 , wherein the milled plant part of b) is suspended in water. 
     
     
         6 . The method of  claim 1 , wherein c) is between about 60° C. to about 100° C. 
     
     
         7 . The method of  claim 1 , wherein the viscosity changes of d) are measured from a range consisting of 10 to 300 seconds. 
     
     
         8 . The method of  claim 1 , wherein viscosity changes of d) are measured at a maximum of 74 seconds. 
     
     
         9 . The method of  claim 1 , wherein the plant part of a) is mixed with negative background. 
     
     
         10 . The method of  claim 1 , wherein the plant part is maize seed 
     
     
         11 . The method of  claim 1 , wherein the endogenous polysaccharide substrate is starch. 
     
     
         12 . The method of  claim 1 , wherein the endogenous polysaccharide is of an unknown concentration. 
     
     
         13 . The method of  claim 1 , wherein said polysaccharide-hydrolyzing enzyme is a alpha-amylase. 
     
     
         14 . The method of  claim 13 , wherein the alpha-amylase is a themotolerant alpha-amylase. 
     
     
         15 . The method of  claim 14 , wherein the alpha-amylase is selected from the group consisting of 797GL3 or D45. 
     
     
         16 . The method of  claim 1 , wherein d) comprises collecting viscosity data from at least two time points. 
     
     
         17 . The method of  claim 1 , wherein the concentration is measured at % admix. 
     
     
         18 . A method of calculating the activity of a polysaccharide-hydrolyzing enzyme, the method comprising the steps of:
 a) milling a plant part wherein the plant part contains an endogenous polysaccharide substrate;   b) suspending the milled plant part in liquid to make a solution wherein the solution comprises a polysaccharide-hydrolyzing enzyme that hydrolyses said endogenous polysaccharide substrate under active conditions;   c) heating the solution to a temperature above a gelatinization temperature of the polysaccharide substrate and wherein the polysaccharide-hydrolyzing enzyme is active;   d) measuring viscosity data of the solution for at least one time point wherein the time point is at least 30 seconds;   e) comparing the viscosity data of d) with a calibration curve wherein the calibration curve is generated in the same manner using at least two calibration samples of known polysaccharide-hydrolyzing enzyme activity and correlates said polysaccharide-hydrolyzing enzyme activity with viscosity;   f) measuring said polysaccharide-hydrolyzing enzyme activity.   
     
     
         19 . The method of  claim 18 , wherein said plant part is a transgenic plant part comprising a heterologous polysaccharide hydrolyzing enzyme. 
     
     
         20 . The method of  claim 18 , wherein b) comprises 1 or more additional polysaccharide-hydrolyzing enzymes 
     
     
         21 . The method of  claim 18 , wherein said polysaccharide-hydrolyzing enzyme is selected from the group consisting of alpha-amylase, cellulase, glucoamylase, beta-amylase, endoglucanase, pullulinase, xylanase and combinations thereof. 
     
     
         22 . The method of  claim 18 , wherein the milled plant part of b) is suspended in water. 
     
     
         23 . The method of  claim 18 , wherein c) is between about 60° C. to about 100° C. 
     
     
         24 . The method of  claim 18 , wherein the viscosity changes of d) are measured from a range consisting of 10 to 300 seconds. 
     
     
         25 . The method of  claim 18 , wherein viscosity changes of d) are measured at a maximum of 74 seconds. 
     
     
         26 . The method of  claim 18 , wherein the plant part of a) is mixed with negative background. 
     
     
         27 . The method of  claim 18 , wherein the plant part is maize seed 
     
     
         28 . The method of  claim 18 , wherein the endogenous polysaccharide substrate is starch. 
     
     
         29 . The method of  claim 18 , wherein said polysaccharide-hydrolyzing enzyme is a alpha-amylase. 
     
     
         30 . The method of  claim 29 , wherein the alpha-amylase is a themotolerant alpha-amylase. 
     
     
         31 . The method of  claim 30 , wherein the alpha-amylase is selected from the group consisting of 797GL3 or D45. 
     
     
         32 . The method of  claim 29 , wherein alpha-amylase activity is greater than about 2 U/g 
     
     
         33 . The method of  claim 18 , wherein d) comprises collecting viscosity data from at least two time points. 
     
     
         34 . A method of measuring a polysaccharide-hydrolyzing enzyme concentration, the method comprising the steps of:
 a) milling a plant part wherein the plant part contains an endogenous polysaccharide substrate;   b) suspending the milled plant part in liquid to make a solution wherein the solution comprises a polysaccharide-hydrolyzing enzyme that hydrolyses said endogenous polysaccharide substrate;   c) heating the solution to a temperature above a gelatinization temperature of the polysaccharide substrate and wherein the polysaccharide-hydrolyzing enzyme is active;   d) measuring glucose levels of the solution for at least one time point wherein the time point is at least 30 seconds;   e) comparing the glucose levels of d) with a calibration curve wherein the calibration curve is generated in the same manner using at least two calibration samples of known polysaccharide-hydrolyzing enzyme concentration and correlates said polysaccharide-hydrolyzing enzyme concentration with glucose levels;   f) measuring said polysaccharide-hydrolyzing enzyme concentration.   
     
     
         35 . A method of calculating the activity of a polysaccharide-hydrolyzing enzyme, the method comprising the steps of:
 a) milling a plant part wherein the plant part contains an endogenous polysaccharide substrate;   b) suspending the milled plant part in liquid to make a solution wherein the solution comprises a polysaccharide-hydrolyzing enzyme that hydrolyses said endogenous polysaccharide substrate under active conditions;   c) heating the solution to a temperature above a gelatinization temperature of the polysaccharide substrate and wherein the polysaccharide-hydrolyzing enzyme is active;   d) measuring glucose levels of the solution for at least one time point wherein the time point is at least 30 seconds;   e) comparing the glucose levels of d) with a calibration curve wherein the calibration curve is generated in the same manner using at least two calibration samples of known polysaccharide-hydrolyzing enzyme activity and correlates said polysaccharide-hydrolyzing enzyme activity with glucose levels;   f) measuring said polysaccharide-hydrolyzing enzyme activity.