Method of measuring alpha amylase
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 an α-amylase concentration, the method comprising the steps of:
a) milling maize seed, wherein the maize seed contains an endogenous starch substrate, wherein said maize seed is transgenic maize seed comprising event 3272 expressing a heterologous thermotolerant 797GL3 α-amylase;
b) suspending the milled maize seed in liquid to make a solution wherein the solution comprises the heterologous thermotolerant 797GL3 α-amylase that hydrolyses said endogenous starch substrate;
c) heating the solution to a temperature above a gelatinization temperature of the endogenous starch substrate and wherein the heterologous thermotolerant 797GL3 α-amylase is active, wherein the temperature is between about 60° C. to about 100° C.;
d) measuring viscosity of the solution for at least two time points from a range consisting of 30 seconds to 2 minutes;
e) comparing the viscosity of d) with a calibration curve wherein the calibration curve is generated in the same manner using at least three calibration samples of known α-amylase concentrations;
f) measuring said heterologous thermotolerant 797GL3 α-amylase concentration from said calibration curve.
2. The method of claim 1 , wherein b) comprises 1 or more additional polysaccharide-hydrolyzing enzymes.
3. The method of claim 1 , wherein the milled maize seed of b) is suspended in water.
4. The method of claim 1 , wherein the endogenous starch substrate is of an unknown concentration.
5. The method of claim 1 , wherein the concentration is measured as % admix of transgenic maize seed comprising event 3272 expressing the heterologous thermotolerant 797GL3 α-amylase.
6. The method of claim 1 , wherein the at least two time points of d) are at least 30 seconds, wherein viscosity changes are measured at a maximum of 74 seconds.