A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates

Journal of Biotechnology
Martin GräberPatrick Adlercreutz

Abstract

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...Continue Reading

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Citations

Sep 6, 2013·Journal of Industrial Microbiology & Biotechnology·Nguyen Huy Thuan, Jae Kyung Sohng
May 29, 2014·Chemistry, an Asian Journal·Wouter M J KloostermanKatja Loos

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