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

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 and 3, respectively, showing a significant difference in their ability to synthesise hexyl-beta-d-glucopyranoside by reversed hydrolysis.

journal_name

J Biotechnol

journal_title

Journal of biotechnology

authors

Gräber M,Andersson M,Rundbäck F,Pozzo T,Karlsson EN,Adlercreutz P

doi

10.1016/j.jbiotec.2009.11.005

subject

Has Abstract

pub_date

2010-01-15 00:00:00

pages

186-92

issue

2

eissn

0168-1656

issn

1873-4863

pii

S0168-1656(09)00504-5

journal_volume

145

pub_type

杂志文章

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