Advanced

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

Gräber, Martin; Andersson, Mats; Rundbäck, Fabian LU ; Pozzo, Tania LU ; Nordberg Karlsson, Eva LU and Adlercreutz, Patrick LU (2010) In Journal of Biotechnology 145. p.186-192
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-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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biotechnology
volume
145
pages
186 - 192
publisher
Elsevier
external identifiers
  • wos:000274445700012
  • pmid:19914310
  • scopus:72949112011
ISSN
1873-4863
DOI
10.1016/j.jbiotec.2009.11.005
language
English
LU publication?
yes
id
a07cc19c-fcc5-4505-9de1-3ac73684764f (old id 1511922)
date added to LUP
2009-12-01 09:58:46
date last changed
2018-05-29 11:39:13
@article{a07cc19c-fcc5-4505-9de1-3ac73684764f,
  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-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.},
  author       = {Gräber, Martin and Andersson, Mats and Rundbäck, Fabian and Pozzo, Tania and Nordberg Karlsson, Eva and Adlercreutz, Patrick},
  issn         = {1873-4863},
  language     = {eng},
  pages        = {186--192},
  publisher    = {Elsevier},
  series       = {Journal of Biotechnology},
  title        = {A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates.},
  url          = {http://dx.doi.org/10.1016/j.jbiotec.2009.11.005},
  volume       = {145},
  year         = {2010},
}