A new method for the determination of trehalose by flow 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-Haën. The immobilization rate is about 60%. The FIA 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).
Copyright 1997 Academic Press.