DETERMINATION OF TREHALOSE BY FLOW-INJECTION ANALYSIS USING IMMOBILIZED TREHALASE

A new method for the determination of trehalose by how injection analy sis (FIA) is described. The basic principle is the hydrolysis of the d isaccharide trehalose into its monomer D-glucose by trehalase, a perip lasmic enzyme of Escherichia coli. D-glucose is quantified spectrophot ometrically after reaction with hexokinase and glucose-6-phosphate deh ydrogenase. Trehalase is prepared by osmotic shock from a recombinant E, coli strain and precipitated with ammonium sulfate. The enzyme is i mmobilized on VA-Epoxy Biosynth from Riedel-de-Haen, The immobilizatio n rate is about 60%. The FlA signals show a nonlinear dependence on th e trehalose concentration. The resulting curve corresponds to a second -order polynomial that serves as a calibration function for test sampl es. Immobilized trehalase was used during a period of 4 months without any loss of suitability. Several samples of fermentation broth were t ested. 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.