An enhanced system to screen trehalase inhibitors using a microplate assay with a housefly enzyme source

Trehalase (EC 3.2.1.28) hydrolyses the non-reducing disaccharide trehalose, the main source of glucose in insects, nematodes, and fungi, into 2 glucose molecules. Therefore, development of specific and potent trehalase inhibitors as control agents against insect pests and other deleterious organisms has been of great interest. For the establishment of a 96-well microplate assay to detect trehalase activity, the o-toluidine method of glucose determination was deliberately modified with whole body extracts from housefly (Musca domestica Linnaeus) larvae serving as an enzyme source. In the microplate trehalase assay, optical density (OD) at 630 nm increased proportionally with acetic acid concentration up to 90%, with the OD rate of increase greatest at 10% o-toluidine. Sensitivity in a 96-well plate reaction was slightly better than with spectrophotometric measurements, especially at low concentrations of glucose. Samples from 70% ammonium sulphate precipitation showed the greatest activity with minimum noise caused by endogenous reducing sugars and a maximum enzymatic activity at pH 4.5 and 40 °C, with substrate inhibition at trehalose concentrations greater than 320.0 mM. The I50 values of the 2 trehalase inhibitors tested, amygdalin and validamycin A, were 31.7 mM and 0.4 mM, respectively. In conclusion, this 96-well microplate assay of trehalase activity using the whole body extract from housefly larvae as an enzyme source might be useful in discovery of potent trehalase inhibitors through the development of new insecticides from screening numerous synthetic and natural compounds.