Miniaturization of hydrolase assays in thermocyclers.

ABSTRACT

We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and ß-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a ß-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.