Change search
Refine search result
1 - 48 of 48
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1. Ahlqvist, J.
    et al.
    Kumar, A.
    Sundstrom, H.
    Ledung, E.
    Hornsten, E. G.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Mattiasson, B.
    Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 2, p. 216-225Article in journal (Refereed)
    Abstract [en]

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B.. 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  • 2.
    Basselet, Pascal
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Wegrzyn, Grzegorz
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)2008In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Background: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. Results: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2x), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes. Conclusion: In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.

  • 3. Bollok, Monika
    et al.
    Henriksson, Hongbin
    Kallas, Åsa
    KTH, Superseded Departments, Biotechnology.
    Jahic, Mehmedalija
    KTH, Superseded Departments, Biotechnology.
    Teeri, Tula
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Production of plant xyloglucan endotransglycosylase (XET) using the methylotrophic yeast Pichia pastorisIn: Applied Biochemistry and Biotechnology, ISSN 0273-2289, E-ISSN 1559-0291Article in journal (Other academic)
  • 4.
    Bollok, Monika
    et al.
    KTH, School of Biotechnology (BIO).
    Henriksson, Hongbin
    KTH, School of Biotechnology (BIO).
    Kallas, Åsa
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris2005In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, Vol. 126, p. 61-77Article in journal (Refereed)
    Abstract [en]

    The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.

  • 5. Castan, A.
    et al.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Characteristics of a DO-controlled fed-batch culture of Escherichia coli2000In: Bioprocess engineering (Berlin. Print), ISSN 0178-515X, E-ISSN 1432-0797, Vol. 22, no 6, p. 509-515Article in journal (Refereed)
    Abstract [en]

    The DO-controlled glucose limited fed-batch technique was investigated in an E. coli process for production of a recombinant protein. The k(L)ac* value (oxygen transfer rate at zero oxygen concentration) was calculated from on-line gas analysis data during the process. In the investigated processes with induced production of recombinant protein, the k(L)ac* value decreased drastically several hours after induction. The reason for the decrease was found in increasing concentrations of DNA in the medium and increased viscosity due to cell lysis. The consequences of such a dramatic decrease in the volumetric oxygen transfer coefficient on the glucose feed and specific rates are described in computer simulations and experimental data.

  • 6. Castan, A.
    et al.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli2002In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 77, no 3, p. 324-328Article in journal (Refereed)
    Abstract [en]

    Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.

  • 7. Castan, A.
    et al.
    Heidrich, J.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    The use of flow cytometry to detect nucleic acids attached to the surface of Escherichia coli in high cell density fed-batch processes2002In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 24, no 3, p. 219-224Article in journal (Refereed)
    Abstract [en]

    E. coli was grown in an aerobic fed-batch process for the production of a recombinant protein (rhGH). The cells were examined by flow cytometry and PI (propidium iodide) staining. The fluorescence of the PI-stained cells increased with increasing concentrations of DNA in the medium. Furthermore, DNA and RNA attached to the cell could partly be degraded with DNase/RNase and the fluorescence decreased. Formate excretion during the aerobic processes may be due to DNA and possibly also RNA attached to the cell surface, so creating diffusion resistance.

  • 8. Castan, A.
    et al.
    Nasman, A.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone2002In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 30, no 7, p. 847-854Article in journal (Refereed)
    Abstract [en]

    In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.

  • 9.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, M.
    Stendahl-Andersen, Helle
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes2005In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 27, no 6, p. 399-406Article in journal (Refereed)
    Abstract [en]

    An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.

  • 10.
    Charoenrat, Theppanya
    et al.
    KTH, School of Biotechnology (BIO).
    Ketudat-Cairns, Mariena
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth2006In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 122, no 1, p. 86-98Article in journal (Refereed)
    Abstract [en]

    Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST 1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

  • 11. Charoenrat, Theppanya
    et al.
    Ketudat-Cairns, Mariena
    Jahic, Mehmedalija
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process2006In: Biochemical engineering journal, ISSN 1369-703X, E-ISSN 1873-295X, Vol. 30, no 2, p. 205-211Article in journal (Refereed)
    Abstract [en]

    Two strategies to increase the productivity of secreted Thai Rosewood beta-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient (K(L)a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g(-1) g(-1)) and high-pressure processes (0.035 g g(-1) g(-1)), compared to 0.022 g g(-1) g(-1) in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total beta-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the beta-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins.

  • 12.
    Enfors, Sven-Olof
    et al.
    KTH, Superseded Departments, Biotechnology.
    Jahic, M.
    Rozkov, A.
    Xu, B.
    Hecker, M.
    Jurgen, B.
    Kruger, E.
    Schweder, T.
    Hamer, G.
    O'Beirne, D.
    Noisommit-Rizzi, N.
    Reuss, M.
    Boone, L.
    Hewitt, C.
    McFarlane, C.
    Nienow, A.
    Kovacs, T.
    Tragardh, C.
    Fuchs, Laszlo
    Revstedt, J.
    Friberg, P. C.
    Hjertager, B.
    Blomsten, G.
    Skogman, H.
    Hjort, S.
    Hoeks, F.
    Lin, H. Y.
    Neubauer, P.
    van der Lans, R.
    Luyben, K.
    Vrabel, P.
    Manelius, A.
    Physiological responses to mixing in large scale bioreactors2001In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 85, no 2, p. 175-185Article in journal (Refereed)
    Abstract [en]

    Escherichia coli fed-batch cultivations at 22 m(3) scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/reassimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

  • 13.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, Superseded Departments, Biotechnology.
    Andresen, Heiko
    KTH, Superseded Departments, Biotechnology.
    Albers, Joerg
    Hintsche, Rainer
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip2004In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 3, p. 2-Article in journal (Refereed)
    Abstract [en]

    Background: Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic microorganisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption. Results: A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes. The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes. The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h. The method was also successful when applied directly to unpurified spore and cell extract samples. The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal. Conclusions: The sensitivity, convenience and specificity of the system have shown its potential. In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest. This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification.

  • 14.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, Superseded Departments, Biotechnology.
    Holmgren, Anders
    KTH, Superseded Departments, Biotechnology.
    Andresen, Heiko
    KTH, Superseded Departments, Biotechnology.
    Barken, K. B.
    Wumpelmann, M.
    Albers, J.
    Hintsche, R.
    Breitenstein, A.
    Neubauer, P.
    Los, M.
    Czyz, A.
    Wegrzyn, G.
    Silfversparre, G.
    Jurgen, B.
    Schweder, T.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Electric chips for rapid detection and quantification of nucleic acids2004In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 19, no 6, p. 537-546Article in journal (Refereed)
    Abstract [en]

    A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the biorecognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

  • 15.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Liu, Yanling
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gene-based identification of bacterial colonies with an electric chip2005In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 345, no 2, p. 270-276Article in journal (Refereed)
    Abstract [en]

    A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.

  • 16.
    Gabig-Ciminska, Magdalena
    et al.
    KTH, Superseded Departments, Biotechnology.
    Los, M.
    Holmgren, Anders
    KTH, Superseded Departments, Fibre and Polymer Technology.
    Albers, J.
    Czyz, A.
    Hintsche, R.
    Wegrzyn, G.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 324, no 1, p. 84-91Article in journal (Refereed)
    Abstract [en]

    Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample.. These analyses confirmed the specificity of the assay.

  • 17. Han, L.
    et al.
    Doverskog, M.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Effect of glycine on the cell yield and growth rate of Escherichia coli: evidence for cell-density-dependent glycine degradation as determined by C-13 NMR spectroscopy2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 92, no 3, p. 237-249Article in journal (Refereed)
    Abstract [en]

    Addition of selected amino acids could be a means to improve production of recombinant proteins in industrial processes. We found that glycine increased the maximum specific growth rate of Escherichia colt from 0.67 to 0.78 h(-1), and the cell yield from 0.57 to 0.98 g dry weight per g substrate, when supplemented to batch cultures in a glucose-mineral medium. Maximum effect occurred at pH 6.8, at a glycine concentration of 6-12 mmol l(-1), and at cell densities below 1.15 g dry weight l(-1) (0D(610)(.)3). When glycine was added to a culture at a cell density of 1.15 g l(-1) or above, no growth promoting effect of glycine was seen. The 'glycine effect' was not due to CO2 produced by the glycine cleavage system (GCV), and the lack of effect at higher cell densities was not masked by acetate accumulation, but coincided with increased acetate production. The metabolism of glycine was further investigated in cultures supplied with [2-C-13] labelled glycine, and the redistribution of label in the [1-C-13], [2-C-13], and [1,2-C-13] isotopomeres of excreted acetate was analysed by C-13 NMR. The NMR data revealed that very little degradation of glycine occurred at cell densities below 1.15 g l(-1). Simultaneously the biosynthesis of serine and glycine was repressed as judged by the absence of [2-C-13] acetate, implying that added glycine was used as a source of glycine, serine, one-carbon units, and threonine. At cell densities above 1.15 g l(-1), 53% of the consumed glycine carbon was excreted as acetate. Degradation of glycine was associated with an increased uptake rate, cleavage by GCV, and degradation of both glycine-derived serine, and glucose-derived serine to pyruvate. This switch in metabolism appears to be regulated by quorum sensing.

  • 18. Han, L.
    et al.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Changes in intracellular metabolite pools, and acetate formation in Escherichia coli are associated with a cell-density-dependent metabolic switch2002In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 24, no 6, p. 483-488Article in journal (Refereed)
    Abstract [en]

    A cell density-dependent metabolic switch in amino acid metabolism occurs in E. coli W3110 batch cultures at 1.15 g dry wt l(-1) (Han L, Doverskog M, Enfors S-O, Haggstrom L, 2002, J. Biotechnol. 92: 237-249). A two- to three-fold decrease of the concentration of most glycolytic and citric acid cycle metabolites, and an increase in acetyl-CoA concentration after the switch, indicates that the central metabolism also is affected. The specific acetate production rate decreases throughout the culture, except for a temporary increase at the switch point. The intracellular acetate concentration remains relatively constant during the culture.

  • 19. Han, L.
    et al.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Häggström, Lena
    KTH, Superseded Departments, Biotechnology.
    Escherichia coli high-cell-density culture: carbon mass balances and release of outer membrane components2003In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 25, no 4, p. 205-212Article in journal (Refereed)
    Abstract [en]

    Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO2 and acetate was similar to90% at the end of the culture (25 h, 27 g L-1 dw). The missing carbon remained as soluble organic compounds in the medium. Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at similar to1 g L-1) contributed to 63% of the extracellular carbon. The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture. Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred. Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the carbon mass. The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.

  • 20.
    Henriksson, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Teeri, Tuula
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Improvements of Pichia pastoris fermentation technique for production of recombinant proteinsManuscript (Other academic)
  • 21. Ignatova, Z.
    et al.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Hobbie, M.
    Taruttis, S.
    Vogt, C.
    Kasche, V.
    The relative importance of intracellular proteolysis and transport on the yield of the periplasmic enzyme penicillin amidase in Escherichia coli2000In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 26, no 04-feb, p. 165-170Article in journal (Refereed)
    Abstract [en]

    Intracellular proteolysis is an important mechanism for regulating the level of the periplasmic enzyme penicillin amidase in Escherichia coli. Evidence is presented that the active enzyme is localized in the periplasmic space and maturation of pro-enzyme occurs during transport through the cytoplasmic membrane or rapidly after its entrance in the periplasm. The rate constants of the transport through cytoplasmic membrane and of the intracellular proteolysis were estimated to be 0.01 h and 0.5 h, respectively. This indicates that more than 90% of the synthesized pre-pro-enzyme is lost by intracellular proteolysis occurring in the cytoplasm.

  • 22. Jahic, M.
    et al.
    Knoblechner, J.
    Charoenrat, T.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Interfacing Pichia pastoris cultivation with expanded bed adsorption2006In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 93, no 6, p. 1040-1049Article in journal (Refereed)
    Abstract [en]

    For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L (.) h.

  • 23.
    Jahic, Mehmedalija
    et al.
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Gustavsson, Malin
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Jansen, Ann-Katrin
    Martinelle, Mats
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology. KTH, Superseded Departments (pre-2005), Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes2003In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 102, no 1, p. 45-53Article in journal (Refereed)
    Abstract [en]

    A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degreesC during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

  • 24.
    Jahic, Mehmedalija
    et al.
    KTH, Superseded Departments, Biotechnology.
    Rotticci-Mulder, Johanna C.
    Martinelle, Mats
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Hult, Karl
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein2002In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 24, no 6, p. 385-393Article in journal (Refereed)
    Abstract [en]

    A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O-2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.

  • 25. Jahic, Mehmedalija
    et al.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Charoenrat, Theppanya
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Process technology for production and recovery of heterologous proteins with Pichia pastoris2006In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 22, no 6, p. 1465-1473Article, review/survey (Refereed)
    Abstract [en]

    Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.

  • 26.
    Jahic, Mehmedalija
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wallberg, Fredrik
    Bollok, Monika
    KTH, Superseded Departments, Biotechnology.
    Garcia, Percival
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures.2003In: Microbial cell factories, ISSN 1475-2859, Vol. 2, no 1, p. 6-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut+ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. RESULTS: A protein concentration of about 1 g L-1 was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L-1 full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40-70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process. CONCLUSION: Three mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity.

  • 27.
    Larsen Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO), Biochemistry.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO), Biochemistry.
    Screening and production of Pseudozyma (Candida) antarctica lipase B in Pichia pastoris using the GAP promoter as alternative to the AOX promoter expression systemManuscript (preprint) (Other academic)
  • 28. Le, T. N.
    et al.
    Do, T. H.
    Nguyen, T. N.
    Tran, N. T.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Truong, N. H.
    Expression and simple purification strategy for the generation of antimicrobial active enterocin P from Enterococcus faecium expressed in Escherichia coli ER25662014In: Iranian Journal of Biotechnology, ISSN 1728-3043, Vol. 12, no 4, p. 16-25, article id e1154Article in journal (Refereed)
    Abstract [en]

    Background: Enterocin-P of Enterococcus faecium P13 (EntP) is of great interest as a food preservative and medicine due to its non-toxicity and broad antimicrobial spectrum at various pH, as well as its excellent thermal stability. However, recombinant production of EntP is still in laboratory because of low productivity and complex purification process. Objectives: In this study, we aimed to develop efficient methods for high-level expression and convenient purification of the recombinant EntP.Materials and Methods: An artificially synthesized gene (entP) of 132 bp encoding mature enterocin P of E. faecium P13 was cloned in plasmid pTWIN1 under the control of an inducible T7lac promoter for expression of fusion protein EntP- Mxe GyrA mini-intein-chitin binding domain (CBD) (abbreviated by EntP-Int-CBD) in E. coli. Recombinant EntP was released from the fusion protein by DTT digestion and cleaned by distilled water and checked for anti-bacterial activity. Results: The fusion protein was highly expressed in insoluble form in E. coli at 37ºC with 0.05 mM IPTG induction. The insoluble fusion protein EntP-Int-CBD was easily prepared by cell sonication and centrifugation to remove soluble con- taminants. The repeat washing steps with Triton X-100 were applied to reduce contaminants. After DTT-induced self- digestion in urea 4 M, the EntP released from the fusion protein was insoluble in water and easier to be separated from sol- uble Int-CBD by centrifugation. The recombinant peptide was soluble in 20% 2-propanol in 0.1% trifluoroacetic acid (TFA) and exhibited strong anti- Listeria monocytogenes and Staphylococcus aureus activities.Conclusions: This study is the first report providing a simple, quick and straight forward procedure for heterologous production of functional and pure Enterocin P without using any chromatography columns in the purification process.

  • 29. Lin, H. Y.
    et al.
    Hoffmann, F.
    Rozkov, Aleksei
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Rinas, U.
    Neubauer, P.
    Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli2004In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 87, no 5, p. 602-613Article in journal (Refereed)
    Abstract [en]

    Guanosine-3'5'-tetraphosphate (ppGpp) and as, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.

  • 30. Lin, H. Y.
    et al.
    Mathiszik, B.
    Xu, B.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Neubauer, P.
    Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli2001In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 73, no 5, p. 347-357Article in journal (Refereed)
    Abstract [en]

    A simple pulse-based method for the determination of the maximum uptake capacities for glucose a nd oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where mu was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when mu declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.

  • 31.
    Liu, Yanling
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Basselet, Pascal
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Los, Marcin
    Wegrzyn, Grzegorz
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Rapid determination of virulence factors in EHEC colonies with an electric DNA chip arrayArticle in journal (Other academic)
  • 32.
    Liu, Yanling
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Elsholz, Bruno
    Fraunhofer Institute for Silicon Technology, Itzehoe, Germany.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Confirmative electric DNA array-based test for food poisoning Bacillus cereus2007In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 70, no 1, p. 55-64Article in journal (Refereed)
    Abstract [en]

    Detection of the full set of toxin encoding genes involved in gastrointestinal diseases caused by B. cereus was performed. Eight genes determining the B. cereus pathogenicity, which results in diarrhea or emesis, were simultaneously evaluated on a 16-position electrical chip microarray. The DNA analyte preparation procedure comprising first 5 min of ultrasonic treatment, DNA extraction, and afterwards an additional 10 min sonication, was established as the most effective way of sample processing. No DNA amplification step prior to the analysis was included. The programmed assay was carried out within 30 min, once the DNA analyte from 10(8) bacterial cells, corresponding to one agar colony, was subjected to the assay. In general, this work represents a mature analytical way for DNA review. It can be used under conditions that require almost immediate results.

  • 33.
    Liu, Yanling
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Elsholz, Bruno
    Fraunhofer Institute for Silicon Technology, Itzehoe, Germany.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria2008In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 382, no 2, p. 77-86Article in journal (Refereed)
    Abstract [en]

    Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals Lip to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease Of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.

  • 34. Rozkov, A.
    et al.
    Schweder, T.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins2000In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 27, no 10, p. 743-748Article in journal (Refereed)
    Abstract [en]

    A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg.g(-1)) compared to the fusion protein SpA-beta galactosidase (138 mg.g(-1)) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the Ist order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg.g(-1) to 120 mg.g(-1). The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg.g(-1) in II h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.

  • 35.
    Rozkov, Aleksei D.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Analysis and control of proteolysis of recombinant proteins in Escherichia coli.2004In: Advances in biochemical engineering/biotechnology, ISSN 0724-6145, Vol. 89, p. 163-195Article in journal (Refereed)
    Abstract [en]

    Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli. Important properties of E. coli proteases, which are relevant for the production of recombinant proteins, are reviewed. Furthermore, various strategies to control the proteolysis of the recombinant proteins are presented. These strategies for control of proteolysis can be applied on various stages of the process: design of more stable protein, a modification of the host cell in respect to proteolytic activity, optimisation of cultivation and downstream processing. However, before implementing these measures the proteolysis rate should be measured in order to calculate a potential benefit of reduced proteolysis rate.

  • 36.
    Råvik, Mattias
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    High-pressure homogenisation of Escherichia coli and its effect on viscosity and DNA fragmentationManuscript (Other academic)
  • 37.
    Sundström, Heléne
    et al.
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    A bioreactor system for high throughput process developmentManuscript (Other academic)
  • 38.
    Sundström, Heléne
    et al.
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Software sensors for fermentation processes2008In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 31, no 2, p. 145-152Article in journal (Refereed)
    Abstract [en]

    Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R-O/S sensor which is the ratio between oxygen and energy substrate consumption. The R-O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R-O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.

  • 39.
    Sundström, Heléne
    et al.
    KTH, Superseded Departments, Biotechnology.
    Wållberg, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Ledung, E.
    Department of Biology and Chemical Engineering, Mälardalen University.
    Norrman, B.
    Department of Biology and Chemical Engineering, Mälardalen University.
    Hewitt, C. J.
    Department of Chemical Engineering (Biochemical Engineering), University of Birmingham.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes2004In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 26, no 19, p. 1533-1539Article in journal (Refereed)
    Abstract [en]

    In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml(-1)) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml(-1) dropped to similar to10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml(-1) probably reflects a loss of cell division capability rather than cell death.

  • 40. Surribas, Anna
    et al.
    Stahn, Rainer
    Montesinos, Jose Luis
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Valero, Francisco
    Jahic, Mehmedalija
    Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 130, no 3, p. 291-299Article in journal (Refereed)
    Abstract [en]

    Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.

  • 41.
    Svensson, Marie
    et al.
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Impact of stirring on the endotoxin release from Escherichia coliManuscript (Other academic)
  • 42.
    Svensson, Marie
    et al.
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Modeling of temperatur-dependent growth of Escherichia coli in a PH-auxostat and temperatur-limited fed-batch cultureArticle in journal (Refereed)
  • 43.
    Svensson, Marie
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Han, Ling
    KTH, School of Biotechnology (BIO).
    Silfversparre, Gustav
    KTH, School of Biotechnology (BIO).
    Häggström, Lena
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-olof
    KTH, School of Biotechnology (BIO).
    Control of endotoxin release in Escherichia coli fed-batch cultures2005In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 27, no 2, p. 91-97Article in journal (Refereed)
    Abstract [en]

    High amounts of outer membrane (OM) components were released in glucose-limited fed-batch (GLFB) cultures at 37 degrees C at specific growth rates approaching 0.05 h(-1). Endotoxin analyses from a 20 degrees C GLFB culture gave similar results. An alternative fermentation technique, the temperature-limited fed-batch (TLFB) technique, reduced the endotoxin concentration in a culture with a biomass concentration of 30 g l(-1) from the 850 mg l(-1) in traditional GLFB cultures to about 20 mg l(-1). The TLFB technique uses the temperature to regulate the dissolved oxygen tension, while all substrate components are unregulated. It appears to be severe glucose limitation that triggers the extensive release of endotoxins rather than a low growth rate. Furthermore, it is not the low temperature that stabilizes the OM when using the TLFB technique. Simulations and experimental data show that this technique results in the same biomass productivity as the GLFB technique.

  • 44.
    Svensson, Marie
    et al.
    KTH, School of Biotechnology (BIO).
    Svensson, Ingrid
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process2005In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 67, no 3, p. 345-350Article in journal (Refereed)
    Abstract [en]

    The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, that occurs in the glucose-limited fed-batch (GLFB) processes at specific growth rates below 0.1 h(-1). The TLFB and the GLFB process were compared to each other when applied to produce the periplasmic, constitutively expressed, enzyme beta-lactamase. The extraction of the enzyme was performed by osmotic shock. A higher production of beta-lactamase was achieved with the TLFB technique while no difference in the endotoxin release was found during the extraction procedure. Furthermore, it was found that growth at declining temperature, generated by the TLFB technique, gradually stabilizes the cytoplasmic membrane, resulting in a significantly increased product quality in the extract from the TLFB cultures in the osmotic shock treatment.

  • 45. Vrabel, P.
    et al.
    van der Lans, Rgjm
    van der Schot, F. N.
    Luyben, Kcam
    Xu, B.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    CMA: integration of fluid dynamics and microbial kinetics in modelling of large-scale fermentations2001In: Chemical Engineering Journal, ISSN 1385-8947, E-ISSN 1873-3212, Vol. 84, no 3, p. 463-474Article in journal (Refereed)
    Abstract [en]

    Transport limitation is regarded as one of the major phenomena leading to process yield reduction in large-scale fermentations. Knowledge of both the fluid dynamics and the microbial kinetics is needed for understanding and describing situations in large-scale production bioreactors. Microbial kinetics of Escherichia coli including flow metabolism was determined in lab-scale batch and fed-batch experiments. The effect of high substrate fluctuations on metabolism was quantified in scale-down experiments. This knowledge was incorporated into a flow model based on the compartment model approach (CMA). The flow model was verified by mixing time experiments on aerated reactors mixed with multiple impellers at different regimes with liquid volumes 8-22 m(3). The integral model, predicting local glucose, acetate and biomass concentrations in different parts of the reactor, was compared to three large-scale fermentations performed in two different reactors. If lab-scale kinetics was used, the biomass prediction overestimated the biomass concentration. Lab-scale kinetics modified by the results of scale-down experiments incorporating the effect of substrate fluctuations gave a rather satisfying description of biomass concentration. Glucose gradients in different parts of the reactor and acetate produced as a result of overflow metabolism were predicted on a qualitative level. The simulations show that at present the decisive factor for a successful integration of fluid dynamics and microbial kinetics is the kinetics.

  • 46.
    Wang, Yong-Bin
    et al.
    KTH, School of Information and Communication Technology (ICT), Integrated Devices and Circuits.
    Assefaw-Redda, Yohannes
    KTH, School of Biotechnology (BIO).
    Gabig-Ciminska, Magdalena
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Östling, Mikael
    KTH, School of Information and Communication Technology (ICT), Microelectronics and Information Technology, IMIT.
    Zhang, Shi-Li
    KTH, School of Information and Communication Technology (ICT), Microelectronics and Information Technology, IMIT.
    A novel dual mode capacitor biosensor for real-time, label-free DNA detection2006In: 2006 INTERNATIONAL ELECTRON DEVICES MEETING, VOLS 1 AND 2, NEW YORK: IEEE , 2006, p. 447-450Conference paper (Refereed)
    Abstract [en]

    A novel biosensor working in both MOS capacitor and electric double-layer capacitor mode for real-time, label-free DNA detection is presented. The former mode measures density and polarity of charged biomolecules to be monitored, while the latter mode appears to reveal more of surface-molecule interactions and offers high sensitivity and easiness for diagnosis. A combined mode-utilization yields optimal complementary information of significance.

  • 47.
    Wittrup, Marianne
    et al.
    KTH, School of Biotechnology (BIO).
    Jahic, Mehmedalija
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO).
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Inducible versus constitutive expression of a lipase in Pichia pastoris: A comparative study using different fermentation techniques2007In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 131, no 2, p. S149-S149Article in journal (Other academic)
  • 48.
    Wållberg, Fredrik
    et al.
    KTH, School of Biotechnology (BIO).
    Sundström, Heléne
    KTH, School of Biotechnology (BIO).
    Ledung, E.
    Department of Biology and Chemical Engineering, Mälardalen University.
    Hewitt, C. J.
    Centre for Formulation Engineering, School of Engineering (Chemical Engineering), University of Birmingham.
    Enfors, Sven-Olof
    KTH, School of Biotechnology (BIO).
    Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry2005In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 27, no 13, p. 919-926Article in journal (Refereed)
    Abstract [en]

    Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6-48 mg PMP/g CDW) whilst highlighting population heterogeneity.

1 - 48 of 48
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf