Methods for detecting and measuring polysaccharide-hydrolyzing enzymes
AssigneeSyngenta Participations AG
InventorsTricia Charlene COSTELLO, Shib SANKAR, Rogerio T.N. PRATA, Hangsik MOON, Xuejun Sean ZHONG, Myoung KIM
Life of this patent
Estimating the term…
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.
Claims13
A method to determine an admix ratio of transgenic corn expressing a heterologous amylase to conventional corn comprising the steps of:
- a) mixing transgenic corn expressing a heterologous amylase to conventional corn to a specific admix level;
- b) milling the corn wherein the corn contains an endogenous polysaccharide substrate;
- c) suspending the milled corn in liquid to make a solution wherein the solution comprises a heterologous amylase enzyme that hydrolyses said endogenous polysaccharide substrate;
- d) heating the solution to a temperature above a gelatinization temperature of the polysaccharide substrate and wherein the heterologous amylase enzyme is active;
- e) measuring viscosity data of the solution for at least one time point wherein the time point is at least 30 seconds;
- f) repeat for different admix level;
- g) comparing the viscosity data of e) with a calibration curve wherein the calibration curve is generated in the same manner using at least two calibration samples of different admix level;
- h) determining the admix level of transgenic corn expressing a heterologous amylase to conventional corn.
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 wherein said plant part is a transgenic plant part comprising a heterologous polysaccharide-hydrolyzing enzyme, wherein said polysaccharide-hydrolyzing enzyme is an alpha-amylase, and wherein the alpha-amylase is a thermotolerant alpha-amylase;
- 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 of the solution for at least one time point wherein the time point is at least 30 seconds, wherein viscosity changes are measured at a maximum of 74 seconds;
- 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 polysaccharide-hydrolyzing enzyme concentration;
- f) measuring said polysaccharide-hydrolyzing enzyme concentration.
Dependencies are inferred from the claim text.