High throughput calorimetry for evaluating enzymatic reactions generating phosphate.
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 synthesise 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.
Hoflack
Lieve
L
Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium. Lieve.Hoflack@UGent.be
De Groeve
Manu
M
Desmet
Tom
T
Van Gerwen
Peter
P
Soetaert
Wim
W
D002151:Calorimetry / Q000379:methods*; D003124:Colorimetry; D005964:Glucosyltransferases / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D015203:Reproducibility of Results
D010710:Phosphates; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase