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The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus

Ramchuran, Santosh LU ; Mateus, Bruno; Holst, Olle LU and Nordberg Karlsson, Eva LU (2005) In FEMS Yeast Research 5(9). p.839-850
Abstract
A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3 g l(-1) xylanase with a maximum activity of 3130 U l(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the... (More)
A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3 g l(-1) xylanase with a maximum activity of 3130 U l(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37 kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T-m) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
FEMS Yeast Research
volume
5
issue
9
pages
839 - 850
publisher
Elsevier
external identifiers
  • wos:000229948700006
  • pmid:15925312
  • scopus:19544375638
ISSN
1567-1364
DOI
10.1016/j.femsyr.2004.12.011
language
English
LU publication?
yes
id
619c4042-3fb8-4d4c-bdc0-02259e78101d (old id 155129)
date added to LUP
2007-07-02 16:54:36
date last changed
2017-07-23 03:52:12
@article{619c4042-3fb8-4d4c-bdc0-02259e78101d,
  abstract     = {A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3 g l(-1) xylanase with a maximum activity of 3130 U l(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37 kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T-m) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.},
  author       = {Ramchuran, Santosh and Mateus, Bruno and Holst, Olle and Nordberg Karlsson, Eva},
  issn         = {1567-1364},
  language     = {eng},
  number       = {9},
  pages        = {839--850},
  publisher    = {Elsevier},
  series       = {FEMS Yeast Research},
  title        = {The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus},
  url          = {http://dx.doi.org/10.1016/j.femsyr.2004.12.011},
  volume       = {5},
  year         = {2005},
}