A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates.
The present work describes the development of a novel direct screening method, assayed in 96-well format, for evaluation of enzymatic alkyl glycoside production in a hexanol-water two-phase system. Alkyl glycosides are surfactants with a range of applications and with good biodegradability and low toxicity. Enzymatic synthesis makes it possible to prepare beta-d-glucopyranosides with high purity. In the developed screening assay, hexyl-beta-d-glucopyranoside was chosen as a model product to be synthesised by reversed hydrolysis in a water-hexanol two-phase system. In a first step the model product is produced by glucosidases expressed in E. coli cells in 96-deep-well plates. After phase separation, the hexyl-beta-d-glucopyranoside in the organic phase is degraded enzymatically and the released glucose detected spectrophotometrically at 405nm utilizing peroxidase/glucose oxidase, and the reagent 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The aqueous phase is used to monitor hydrolysis of p-NPG at 405nm, allowing use of a ratio of the two assays to compensate for expression differences. The complete method was used for comparison of two different beta-glucosidases, classified under glycoside hydrolase family 1 and 3, respectively, showing a significant difference in their ability to synthesise hexyl-beta-d-glucopyranoside by reversed hydrolysis.
Gräber
Martin
M
Department of Biotechnology, Lund University, Getingevägen 60, P.O. Box 124, SE-221 00 Lund, Sweden.
Andersson
Mats
M
Rundbäck
Fabian
F
Pozzo
Tania
T
Karlsson
Eva Nordberg
EN
Adlercreutz
Patrick
P
D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005959:Glucosidases / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
D005960:Glucosides; D005959:Glucosidases