This platform consists of a purpose-made robot that grinds, formats, and dispenses precise amounts of solids into 96-well plates, and a liquid-handling station specifically designed to carry out pretreatments, hydrolysis, and the determination of released reducing sugar equivalents using a colorimetric assay.
The determination of released phosphate was performed with the EnzCheck Phosphatase Assay kit (Invitrogen, CA) according to manufacturer guidelines.
Cellulase activity was estimated by determination of released reducing sugars, after incubation of samples (5 g fresh weight) with carboxymethyl cellulose sodium salt (0.7%) for 24 hours at 50°C, in a Bio-Rad Microplate Reader at 690 nm [45].
Colorimetric determination of released inorganic phosphate (Pi) was then conducted in serial samples.
The starch fraction was hydrolyzed with amylase (E.C.3.2.1.1) and amyloglucosidase (E.C.3.2.1.3), and assayed by enzymatic determination of released glucose [ 60].
To avoid release of CPT during sample manipulation, the aliquot of plasma used for the determination of released CPT was immediately acidified.
GENPLAT utilizes individual purified enzymes, statistical experimental design, robotic pipetting of slurries and enzymes, and automated colorimetric determination of released sugars [ 4, 5].
Automated determination of released reducing sugar after hydrolysis was performed using 3-methyl-2-benzothiazolinone hydrozone, as previously described and established [ 52, 53].
However, varying weather conditions throughout the year and the exact determination of the release point on the plot plan and the release elevation are problematic; these issues cause the results to be non-exact and non-integrated.
Hydrolysis of proteins was measured by the determination of the release of a-amino groups directly in milk by the trinitrobenzenesulfonic acid (TNBS) method (Polychroniadou 1988), in which free amino groups react with the TNBS reagent (Sigma-Aldrich, Bornem, Belgium) at pH 9.2 in the absence of light.
