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:
https://lup.lub.lu.se/record/1511922
- author
- Gräber, Martin
; Andersson, Mats
; Rundbäck, Fabian
LU
; Pozzo, Tania
LU
; Nordberg Karlsson, Eva
LU
and Adlercreutz, Patrick
LU
- organization
- publishing date
- 2010
- 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
- pmid:19914310
- 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
- 2016-04-01 10:45:48
- date last changed
- 2022-01-26 02:15:25
@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}},
doi = {{10.1016/j.jbiotec.2009.11.005}},
volume = {{145}},
year = {{2010}},
}