METHODS FOR DETECTING AND MEASURING POLYSACCHARIDE-HYDROLYZING ENZYMES
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.