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.

Description

RELATED APPLICATIONS[0001]The following application claims priority to U.S. Provisional Application No. 61 / 161,182; filed Mar. 18, 2009FIELD OF THE INVENTION[0002]The invention relates generally to methods of detecting and measuring a polysaccharide-hydrolyzing enzyme and more particularly to methods of detecting and measuring the polysaccharide-hydrolyzing enzyme by observing changes in chemical and physical properties of a polysaccharide-containing sample at a pre-determined temperature over a pre-determined time period.BACKGROUND OF THE INVENTION[0003]Polysaccharides, such as starches, are complex carbohydrates composed of monosaccharides joined via glycosidic bonds. They are typically amorphous and insoluble in water. Examples of polysaccharides include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin.[0004]Starch is a major carbohydrate reserve in plant tubers and seed endosperm. The largest source of starch is cor...

AI Technical Summary

Benefits of technology

[0008]Compositions and methods are provided for measuring a polysaccharide-hydrolyzing enzyme in a sample, particularly in a recombinant plant sample. The methods involve measuring changes in chemical and physical properties of the sample, such as viscosity, to detect and / or measure a polysaccharide-hydrolyzing enzyme in the sample. The viscosity changes result from activity of the polysaccharide-hydrolyzing enzyme in degrading polysaccharides in the sample. In particular, the methods involve measuring viscosity of a sample at a pre-determined temperature over a pre-determined time period using endogenous starch at unknown concentrations as a substrate. These methods provide a rapid and reliable assay.

Problems solved by technology

Current methods for detecting and measuring transgenic plant material containing a polysaccharide-hydrolyzing enzyme are time consuming in that they require milling the plant material, extracting the polysaccharide-hydrolyzing enzyme from a plant sample, adding exogenous polysaccharide as a substrate and / or hydrolyzing the polysaccharide for lengthy periods of time.

For example, insufficient α-amylase activity in transgenic corn flour for ethanol production may result in poor ethanol yield in that too little enzyme is available to actively liquefy the starch.

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