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

Santosh Ramchuran

Research output: ThesisDoctoral Thesis (compilation)

202 Downloads (Pure)

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.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biotechnology
Supervisors/Advisors
  • Holst, Olle, Supervisor
Award date2005 Apr 7
Publisher
Print ISBNs91-89627-29-6
Publication statusPublished - 2005

Bibliographical note

Defence details

Date: 2005-04-07
Time: 10:30
Place: Lecture hall A, Center for Chemistry and Chemical Engineering, Getingevägen 60, Lund Institute of Technology

External reviewer(s)

Name: Enfors, Sven-Olof
Title: Professor
Affiliation: Royal Institute of Technology, Stockholm

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<div class="article_info">Eva Nordberg Karlsson, Maher Abou Hachem, Santosh Ramchuran, Hugo Costa, Olle Holst, Åsa Fex Svenningsen and Gudmundur O. Hreggvidsson. <span class="article_issue_date">2004</span>. <a href="javascript:downloadFile(544478)" class="article_link">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</a> <span class="journal_series_title">FEMS microbiology letters</span>, <span class="journal_volume">vol 241</span> <span class="journal_pages">pp 233–242</span>. <span class="journal_distributor">Elsevier</span></div>
<div class="article_info">Santosh O Ramchuran, Olle Holst and Eva Nordberg Karlsson. <span class="article_issue_date">2005</span>. <a href="javascript:downloadFile(544479)" class="article_link">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</a> <span class="journal_series_title">Journal of bioscience and bioengineering</span>, <span class="journal_distributor">Elsevier</span> (inpress)</div>
<div class="article_info">Santosh O Ramchuran, Bruno Mateus, Olle Holst and Eva Nordberg Karlsson. <span class="article_issue_date">2005</span>. <a href="javascript:downloadFile(544480)" class="article_link">The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus</a> <span class="journal_series_title">FEMS yeast research</span>, <span class="journal_distributor">Elsevier</span> (inpress)</div>
<div class="article_info">Eva Nordberg Karlsson, Santosh O. Ramchuran, Pernilla Turner, Lena de Maré, Christian Cimander, Stephane Velut, Åsa Ekman, Christina Wennerberg, Per Hagander and Olle Holst. <span class="article_issue_date"></span>. <span class="article_title">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</span> <span class="journal_series_title">Journal of biotechnology</span>, <span class="journal_distributor">Elsevier</span> (submitted)</div>
<div class="article_info">S. O. Ramchuran, E. Nordberg Karlsson, S. Velut, L. de Maré, P. Hagander and O. Holst. <span class="article_issue_date">2002</span>. <span class="article_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)</span> <span class="journal_series_title">Applied microbiology and biotechnology</span>, <span class="journal_volume">vol 60</span> <span class="journal_pages">pp 408-416</span>. <span class="journal_distributor">Springer</span></div>

Subject classification (UKÄ)

  • Industrial Biotechnology

Keywords

  • Glycoside hydrolase
  • Heterologous protein
  • High cell density cultivation Expression systems
  • Bioteknik
  • Biotechnology
  • Fed-batch

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