| 1. |
- de Maré, Lena, et al.
(author)
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Temperature limited fed-batch cultivation with a probing feeding strategy for Escherichia coli
- 2004
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In: Computer Applications in Biotechnology 2004. - 008044251X ; , s. 73-79
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Conference paper (peer-reviewed)abstract
- A cultivation strategy combining the advantages of temperature limitedfed-batch and probing control is presented.This is a suitable way to produce recombinant proteins while minimizing therelease of endotoxins which complicates the downstream processing.The downside with the temperature limited fed-batch technique has been the difficulty to achieve a controlled excess of glucose inthe reactor without the accumulation of acetic acid when usingstandard sensors.A method using a probing feeding strategy withdown-pulses is here suggested. The technique is investigated insimulations and experiments.
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| 2. |
- Nordberg Karlsson, Eva, et al.
(author)
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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
- 2004
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In: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 241:2, s. 233-242
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Journal article (peer-reviewed)abstract
- Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.
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| 3. |
- Ramchuran, Santosh
(author)
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Bench-Scale Production of Heterologous Proteins from Extremophiles- Escherichia coli and Pichia pastoris based expression systems
- 2005
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Doctoral thesis (other academic/artistic)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.
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| 4. |
- Ramchuran, Santosh, et al.
(author)
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Effect of postinduction nutrient feed composition and use of lactose as inducer during production of thermostable xylanase in Escherichia coli glucose-limited fed-batch cultivations
- 2005
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In: Journal of Bioscience and Bioengineering. - : Elsevier BV. - 1347-4421 .- 1389-1723. ; 99:5, s. 477-484
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Journal article (peer-reviewed)abstract
- Escherichia coli is a microorganism routinely used in the production of heterologous proteins. The overexpression of a xylanase (Xyn10A Delta NC), which originated from the thermophile Rhodo-thermus marinus cloned under the control of the strong T7/lac promoter in a defined medium (mAT) using a substrate-limited feed strategy, was however shown to impose a significant metabolic burden on host cells. This resulted in a decreased cell growth rate and ultimately also a decreased target protein production. The investigation hence centers on the effect of some selected nutrient feed additives (amino acid [Cys] or TCA-intermediates [citrate, succinate, malate]) used to relieve the metabolic burden imposed during the feeding and postinduction phases of these glucose-limited fed-batch cultivations. The use of either succinic acid or malic acid as feed-additives resulted in an increase in production of approximately 40% of the heterologous thermostable xylanase. Furthermore, use of lactose as an alternative inducer of the T7/lac promoter was also proven to be a suitable strategy that significantly prolonged the heterologous protein production phase as compared with induction using isopropyl P-D-thiogalactopyranoside (IPTG).
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| 5. |
- Ramchuran, Santosh, et al.
(author)
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Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris
- 2006
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 71:4, s. 463-472
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Journal article (peer-reviewed)abstract
- A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZ alpha B vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (alpha-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a "batch-induced" cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.
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| 6. |
- Ramchuran, Santosh, et al.
(author)
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Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3)
- 2002
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 60:4, s. 408-416
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Journal article (peer-reviewed)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.
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| 7. |
- Ramchuran, Santosh, et al.
(author)
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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
- 2005
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In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 5:9, s. 839-850
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Journal article (peer-reviewed)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.
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