Abstract

A calorimetric assay is described for the high throughput screening of enzymes that produce inorganic phosphate. In the current example, cellobiose phosphorylase (EC 2.4.1.20) is tested for its ability to synthesize rare disaccharides. The generated phosphate is measured in a high throughput calorimeter by coupling the reaction to pyruvate oxidase and catalase. This procedure allows for the simultaneous analysis of 48 reactions in microtiter plate format and has been validated by comparison with a colorimetric phosphate assay. The proposed assay has a coefficient of variation of 3.14% and is useful for screening enzyme libraries for enhanced activity and substrate libraries for enzyme promiscuity.

Keywords: Calorimetry, high throughput screening, cellobiose phosphorylase, phosphate assay, pyruvate oxidase