A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates.
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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-ss-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-ss-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 ss-glucosidases, classified under glycoside hydrolase family 1 and 3, respectively, showing a significant difference in their ability to synthesise hexyl-ss-D-glucopyranoside by reversed hydrolysis.