TY - JOUR

T1 - 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

AU - Kadow, S.

AU - Betiku, E.

AU - Rinas, U.

AU - Bilitewski, U.

PY - 2005/6/15

Y1 - 2005/6/15

N2 - 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 μM 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. constrains 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.

AB - 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 μM 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. constrains 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.

KW - Amperometric detection

KW - Dextransucrase

KW - Enzyme activity

KW - Fructose dehydrogenase

KW - Glucosyltransferase

KW - Recombinant GTF-S

KW - Screen-printed electrode

UR - http://www.scopus.com/inward/record.url?scp=23844506852&partnerID=8YFLogxK

U2 - 10.1002/bit.20501

DO - 10.1002/bit.20501

M3 - Article

C2 - 15915514

AN - SCOPUS:23844506852

VL - 91

SP - 154

EP - 161

JO - Biotechnology and bioengineering

JF - Biotechnology and bioengineering

SN - 0006-3592

IS - 2

ER -