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US 10,196,669 B2Granted
Method of measuring alpha amylase
AssigneeSyngenta Participations AG
InventorsTricia Charlene Costello, Shib Sankar Basu, Rogerio T. N. Prata, Hangsik Moon, Xuejun Sean Zhong, Myoung Kim
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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.
Claims6
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.
3 more elements
Dependencies are inferred from the claim text.