Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3)
Ramchuran, Santosh LU ; Nordberg Karlsson, Eva LU
; Velut, Stéphane
LU
; de Maré, Lena
LU
; Hagander, Per
LU
and Holst, Olle
LU
(2002)
In Applied Microbiology and Biotechnology
60(4).
p.408-416
- Abstract
- Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production, and the presence of host cell proteases is related to: i) IPTG induction, ii) cell mass concentration at the time of induction and iii) the presence of metabolites (glutamic acid or those from TSB) during the post induction phase of high-cell-density (HCD) fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7000 and 21000 U / (g cdw)], was observed under... (More)
- Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production, and the presence of host cell proteases is related to: i) IPTG induction, ii) cell mass concentration at the time of induction and iii) the presence of metabolites (glutamic acid or those from TSB) during the post induction phase of high-cell-density (HCD) fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7000 and 21000 U / (g cdw)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell mass concentration of 40 g/L), as well as in induced cultivations employing metabolite supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U / (g cdw)] remained relatively unaffected in all cases. We have established that detectable host cell proteases was not the primary reason for the post-induction activity decrease observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/161991
- author
- Ramchuran, Santosh
LU
; Nordberg Karlsson, Eva
LU
; Velut, Stéphane
LU
; de Maré, Lena
LU
; Hagander, Per
LU
and Holst, Olle
LU
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- pulse-feed strategy, xylanase, cellulase
- in
- Applied Microbiology and Biotechnology
- volume
- 60
- issue
- 4
- pages
- 408 - 416
- publisher
- Springer
- external identifiers
-
- wos:000180027700006
- pmid:12466880
- scopus:0037213598
- ISSN
- 1432-0614
- DOI
- 10.1007/s00253-002-1132-3
- language
- English
- LU publication?
- yes
- id
- 569d6702-1354-4b3b-8b97-3c11c9807854 (old id 161991)
- date added to LUP
- 2016-04-01 16:48:47
- date last changed
- 2022-03-15 03:06:04
@article{569d6702-1354-4b3b-8b97-3c11c9807854,
abstract = {{Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production, and the presence of host cell proteases is related to: i) IPTG induction, ii) cell mass concentration at the time of induction and iii) the presence of metabolites (glutamic acid or those from TSB) during the post induction phase of high-cell-density (HCD) fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7000 and 21000 U / (g cdw)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell mass concentration of 40 g/L), as well as in induced cultivations employing metabolite supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U / (g cdw)] remained relatively unaffected in all cases. We have established that detectable host cell proteases was not the primary reason for the post-induction activity decrease observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.}},
author = {{Ramchuran, Santosh and Nordberg Karlsson, Eva and Velut, Stéphane and de Maré, Lena and Hagander, Per and Holst, Olle}},
issn = {{1432-0614}},
keywords = {{pulse-feed strategy; xylanase; cellulase}},
language = {{eng}},
number = {{4}},
pages = {{408--416}},
publisher = {{Springer}},
series = {{Applied Microbiology and Biotechnology}},
title = {{Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3)}},
url = {{http://dx.doi.org/10.1007/s00253-002-1132-3}},
doi = {{10.1007/s00253-002-1132-3}},
volume = {{60}},
year = {{2002}},
}