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

Summary, in English

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.

• Författare: Martin Gräber; Mats Andersson; Fabian Rundbäck; Tania Pozzo; Eva Nordberg Karlsson; Patrick Adlercreutz
• Avdelning/ar: Bioteknik
• Publiceringsår: 2010
• Språk: Engelska
• Sidor: 186-192
• Publikation/Tidskrift/Serie: Journal of Biotechnology
• Volym: 145
• Länkar: http://dx.doi.org/10.1016/j.jbiotec.2009.11.005
• Dokumenttyp: Artikel i tidskrift
• Förlag: Elsevier
• Ämne: Industrial Biotechnology
• Status: Published
• ISBN/ISSN/Övrigt: ISSN: 1873-4863