Determination of trehalose by flow injection analysis using immobilized trehalase.
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).
Meyer zu Düttingdorf
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HD
Institut für Physiologische Chemie, Tierärztliche Hochschule, Bünteweg 17, Hannover, 30559, Germany. biochem@biochemie-tiho-hannover.de
Bachmann
B
B
Buchholz
M
M
Leuchtenberger
W
W
D001322:Autoanalysis / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007202:Indicators and Reagents; D007700:Kinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods; D013816:Thermodynamics; D014198:Trehalase / Q000378:metabolism; D014199:Trehalose / Q000032:analysis*
D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D014199:Trehalose; D014198:Trehalase