Determination of trehalose by flow injection analysis using immobilized trehalase

A new method for the determination of trehalose by how injection analysis (FIA) is described. The basic principle is the hydrolysis of the disaccharide trehalose into its monomer D-glucose by trehalase, a periplasmic enzyme of Escherichia coli. D-glucose is quantified spectrophotometrically after reaction with hexokinase and glucose-6-phosphate dehydrogenase. Trehalase is prepared by osmotic shock from a recombinant E, coli strain and precipitated with ammonium sulfate. The enzyme is immobilized on VA-Epoxy Biosynth from Riedel-de-Haen, The immobilization rate is about 60%. The FlA signals show a nonlinear dependence on the trehalose concentration. The resulting curve corresponds to a second-order polynomial that serves as a calibration function for test samples. Immobilized trehalase was used during a period of 4 months without any loss of suitability. Several samples of fermentation broth were tested. The results are verified by HPLC. Within an interval of 2 to 10 g/L trehalose the recovery is about 100-120% with a precision of 7% (coefficient of variation). (C) 1997 Academic Press.