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Bench-Scale Production of Heterologous Proteins from Extremophiles- Escherichia coli and Pichia pastoris based expression systems

Ramchuran, Santosh LU (2005)
Abstract
Over the past few years considerable research attention has been assigned to extremophiles as sources of extremozymes due to their applicability in industrial processes, and the development of eco-friendly technologies. The establishment of efficient production strategies for heterologous proteins is an empirical process requiring broad background knowledge on available expression systems together with their major advantages and shortcomings. The studies conducted during the course of this thesis has included four enzymes originating from thermophiles namely, thermostable glycoside hydrolases, xylanase and cellulase from Rhodothermus marinus, cyclomaltodextrinase from Anoxybacillus flavithermus and a phospholipase from alkaliphilic... (More)
Over the past few years considerable research attention has been assigned to extremophiles as sources of extremozymes due to their applicability in industrial processes, and the development of eco-friendly technologies. The establishment of efficient production strategies for heterologous proteins is an empirical process requiring broad background knowledge on available expression systems together with their major advantages and shortcomings. The studies conducted during the course of this thesis has included four enzymes originating from thermophiles namely, thermostable glycoside hydrolases, xylanase and cellulase from Rhodothermus marinus, cyclomaltodextrinase from Anoxybacillus flavithermus and a phospholipase from alkaliphilic Bacillus halodurans. Batch cultivation of R. marinus in the presence of xylan allowed low production of the native xylanase in sufficient amounts to probe cell-attachment studies by enzymatic and immunological techniques. Higher levels of the target proteins were achieved by intracellular and extracellular heterologous production using an Escherichia coli and Pichia pastoris based expression system respectively. The production of a functional enzyme is intimately related to the host's cellular machinery furthermore, as a prerequisite, the establishment of efficient bioprocess strategies is crucial for attaining optimum enzyme production yields. The results presented include bench-scale production strategies employing high cell density fed-batch cultivations with E. coli as a host. Also efficient extracellular production of thermostable xylanase and alkaliphilic phospholipase production using the methylotrophic yeast P. pastoris as a host is reported. (Less)
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author
supervisor
opponent
  • Professor Enfors, Sven-Olof, Royal Institute of Technology, Stockholm
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Glycoside hydrolase, Heterologous protein, High cell density cultivation Expression systems, Bioteknik, Biotechnology, Fed-batch
pages
222 pages
publisher
Santosh O. Ramchuran, Biotechnology (LTH), Lund University
defense location
Lecture hall A, Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund Institute of Technology
defense date
2005-04-07 10:30:00
external identifiers
  • other:ISRN: LUTKDH/TKBT--05/1082--SE
ISBN
91-89627-29-6
language
English
LU publication?
yes
additional info
Eva Nordberg Karlsson, Maher Abou Hachem, Santosh Ramchuran, Hugo Costa, Olle Holst, Åsa Fex Svenningsen and Gudmundur O. Hreggvidsson. 2004. The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes FEMS microbiology letters, vol 241 pp 233–242. Elsevier
Santosh O Ramchuran, Olle Holst and Eva Nordberg Karlsson. 2005. Effect of post-induction nutrient feed composition and the use of lactose as inducer during production of thermostable xylanase in Escherichia coli glucose limited fed-batch cultivations Journal of bioscience and bioengineering, Elsevier (inpress)
Santosh O Ramchuran, Bruno Mateus, Olle Holst and Eva Nordberg Karlsson. 2005. The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus FEMS yeast research, Elsevier (inpress)
Eva Nordberg Karlsson, Santosh O. Ramchuran, Pernilla Turner, Lena de Maré, Christian Cimander, Stephane Velut, Åsa Ekman, Christina Wennerberg, Per Hagander and Olle Holst. . Production of two glycoside hydrolases of thermophilic origin in E. coli and analysis of released endotoxins using substrate limited and temperature limited fed-batch cultivation strategies Journal of biotechnology, Elsevier (submitted)
S. O. Ramchuran, E. Nordberg Karlsson, S. Velut, L. de Maré, P. Hagander and O. Holst. 2002. Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3) Applied microbiology and biotechnology, vol 60 pp 408-416. Springer
id
fde63c16-4274-48ab-abbf-bacab79d6e45 (old id 544473)
date added to LUP
2016-04-04 10:38:17
date last changed
2018-11-21 20:59:55
@phdthesis{fde63c16-4274-48ab-abbf-bacab79d6e45,
  abstract     = {{Over the past few years considerable research attention has been assigned to extremophiles as sources of extremozymes due to their applicability in industrial processes, and the development of eco-friendly technologies. The establishment of efficient production strategies for heterologous proteins is an empirical process requiring broad background knowledge on available expression systems together with their major advantages and shortcomings. The studies conducted during the course of this thesis has included four enzymes originating from thermophiles namely, thermostable glycoside hydrolases, xylanase and cellulase from Rhodothermus marinus, cyclomaltodextrinase from Anoxybacillus flavithermus and a phospholipase from alkaliphilic Bacillus halodurans. Batch cultivation of R. marinus in the presence of xylan allowed low production of the native xylanase in sufficient amounts to probe cell-attachment studies by enzymatic and immunological techniques. Higher levels of the target proteins were achieved by intracellular and extracellular heterologous production using an Escherichia coli and Pichia pastoris based expression system respectively. The production of a functional enzyme is intimately related to the host's cellular machinery furthermore, as a prerequisite, the establishment of efficient bioprocess strategies is crucial for attaining optimum enzyme production yields. The results presented include bench-scale production strategies employing high cell density fed-batch cultivations with E. coli as a host. Also efficient extracellular production of thermostable xylanase and alkaliphilic phospholipase production using the methylotrophic yeast P. pastoris as a host is reported.}},
  author       = {{Ramchuran, Santosh}},
  isbn         = {{91-89627-29-6}},
  keywords     = {{Glycoside hydrolase; Heterologous protein; High cell density cultivation Expression systems; Bioteknik; Biotechnology; Fed-batch}},
  language     = {{eng}},
  publisher    = {{Santosh O. Ramchuran, Biotechnology (LTH), Lund University}},
  school       = {{Lund University}},
  title        = {{Bench-Scale Production of Heterologous Proteins from Extremophiles- Escherichia coli and Pichia pastoris based expression systems}},
  url          = {{https://lup.lub.lu.se/search/files/5585860/544478.pdf}},
  year         = {{2005}},
}