Development of a rapid, quantitative glucosyltransferase assay based on a screen-printed fructose enzyme electrode and application to optimization studies on gtfD expression in recombinant Escherichia coli.
A biosensor for fructose determination was used as basis of an assay for the determination of glucosyltransferase (GTF) activities and applied to monitoring recombinant enzyme production. GTFs catalyze the synthesis of glucans from sucrose leading to the release of fructose. Specific fructose determinations in the microM concentration range were achieved with a fructose electrode based on fructose dehydrogenase, which was immobilized on a screen-printed platinum electrode. This electrode was used as basis of the new assay for GTF activity determinations. Depending on the amount of enzyme, the assay was completed within 15-30 min compared to 1-2 h for the traditional photometric assay. From the amount of fructose released in a given reaction time, GTF activities were determined down to approx. 20 U/L. Even unpurified samples from a recombinant GTF-S production process could be analyzed without any problems, and a good correlation was obtained to data obtained from the photometric assay. Analysis of samples from cultures of various rGTF-S-producing recombinant E. coli strains grown on different media with SDS-PAGE and with the new assay identified the same strain and culture medium as optimum for recombinant GTF-S production.
Kadow
S
S
Department of Natural Product Biology, National Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Betiku
E
E
Rinas
U
U
Bilitewski
U
U
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D003199:Computer Systems; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005964:Glucosyltransferases / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis
D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C028761:D-fructose 5-dehydrogenase; D005964:Glucosyltransferases