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  • 1.
    Bandmann, Nina
    et al.
    KTH, Superseded Departments, Biotechnology.
    Collet, Eric
    KTH, Superseded Departments, Biotechnology.
    Leijen, J
    Uhlén, M
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems2000In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 79, no 2, p. 161-172Article in journal (Refereed)
    Abstract [en]

    The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants. Partitioning experiments performed in a PEG 4000/sodium phosphate aqueous two-phase system showed that for variants containing either (WP)(2) or (WP)(4) peptide extensions, 10- to 70-fold increases in the partitioning to the PEG rich top-phase were obtained, when compared to the wild type enzyme. An increased partitioning was also seen for cutinase variants tagged with both tryptophans and charged amino acids, whereas the effect of solely charged peptide extensions was relatively small. In addition, when performing partitioning experiments from cell disintegrates, the (WP)(4)-tagged cutinase showed a similarly high PEG-phase partitioning, indicating that the effect from the peptide tag was unaffected by the background of the host proteins. Taken together, the results show that the partitioning of the recombinantly produced cutinase model enzyme could be significantly improved by relatively minor genetic engineering and that the effects observed for purified proteins are retained also in an authentic whole cell disintegrate system. The results presented should be of general interest also for the improvement of the partitioning properties of other industrially interesting proteins including bulk enzymes.

  • 2.
    Bandmann, Nina
    et al.
    KTH, Superseded Departments, Biotechnology.
    van Alstine, James
    KTH, Superseded Departments, Biotechnology.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins2002In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 93, no 1, p. 1-14Article in journal (Refereed)
    Abstract [en]

    Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated, Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M 13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.

  • 3. Berggren, K.
    et al.
    Tjerneld, F.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Peptide fusion tags with tryptophan and charged residues for control of protein partitioning in PEG-potassium phosphate aqueous two-phase systems2000In: Bioseparation (Dordrecht), ISSN 0923-179X, E-ISSN 1573-8272, Vol. 9, no 2, p. 69-80Article in journal (Refereed)
    Abstract [en]

    A partition study with peptides and recombinant proteins in poly(ethylene glycol)4000-potassium phosphate aqueous two-phase systems has been performed. The aim was to study to what extent the insertion of charged residues could affect protein partition in addition to the already observed effects of tryptophan residues. The model proteins used are based on a staphylococcal protein A derivative, Z, and modified by the insertion of peptide tags close to the C-terminus. The tags differed with respect to their content of both Trp, negatively (Asp) and positively charged (Lys) amino acid residues. The same partitioning trends were observed for the peptides and fusion proteins. The effect of Trp residues was to direct the partitioning towards the PEG phase. The insertion of two negatively charged (Asp) residues into a Trp(4)-tag enhanced the partition towards the PEG phase even more. The introduction of positively charged (Lys) residues in addition to Trp residues, on the other hand, pulled the peptide or protein towards the potassium phosphate phase. The partitioning of peptides gave a good qualitative picture of the effect of the peptide on partitioning when fused to the protein. The efficiencies of the tags were calculated based on partitioning of tags and fusion proteins, and tag efficiencies generally varied between 60 and 85%.

  • 4. Bylund, F.
    et al.
    Castan, A.
    Mikkola, R.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Larsson, G.
    Influence of scale-up on the quality of recombinant human growth hormone2000In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 69, no 2, p. 119-128Article in journal (Refereed)
    Abstract [en]

    The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.

  • 5.
    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.

  • 6. 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.

  • 7. Collen, A.
    et al.
    Penttila, M.
    Stalbrand, H.
    Tjerneld, F.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)phosphate aqueous two-phase system2002In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 943, no 1, p. 55-62Article in journal (Refereed)
    Abstract [en]

    Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)(2), (WP)(4) or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)(4) extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)(4) fused to the catalytic module and a short sequence of the linker [EGI(core-P5)(WP)(4)] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)(4) tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate.

  • 8. 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.

  • 9. 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.

  • 10. Kepka, C.
    et al.
    Collet, E.
    Persson, J.
    Stahl, A.
    Lagerstedt, T.
    Tjerneld, F.
    Veide, Andres
    KTH, Superseded Departments, Biotechnology.
    Pilot-scale extraction of an intracellular recombinant cutinase from E-coli cell homogenate using a thermoseparating aqueous two-phase system2003In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 103, no 2, p. 165-181Article in journal (Refereed)
    Abstract [en]

    A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 1 to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO50PO50 (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)4 tag was used for enhanced target protein partitioning to the EO50PO50 phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO50PO50 phase from the first step was supplemented with a non-ionic detergent (C12-18EO5) and heated to the cloud point (CP) temperature (45 degreesC). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.

  • 11. Kepka, C.
    et al.
    Collet, E.
    Roos, F.
    Tjerneld, F.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography2005In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1075, no 02-jan, p. 33-41Article in journal (Refereed)
    Abstract [en]

    In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)(4) was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to > 95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process. © 2005 Elsevier B.V. All rights reserved.

  • 12.
    Periyannan Rajeswari, Prem Kumar
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101). KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ramachandraiah, Harisha
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Hansson, Jonas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ardabili, Sahar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Russom, Aman
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Development of microfluidic aqueous two-phase system for continuous partitioning of E. coli strains2011In: 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2011, p. 1329-1331Conference paper (Refereed)
    Abstract [en]

    The interaction of bacterial cells with surrounding environment depends on its surface characteristics such as hydrophobicity, hydrophilicity balance and net charge. In this paper, aqueous two-phase system partitioning of Escherichia coli strains based on their difference in surface properties is introduced in a microfluidic system. While aqueous two-phase system is widely use to separate biomolecules on macroscale, the method has not been adapted in microfluidic system. The bacterial cells are partitioned based on their affinity for streams formed by aqueous polymers polyethylene glycol (PEG) and dextran (Dex). Partitioning efficiency of two Escherichia coli strains is currently being optimized.

  • 13. Rex, E.
    et al.
    Rosander, Erica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Røyne, F.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Ulmanen, J.
    A systems perspective on chemical production from mixed food waste: The case of bio-succinate in Sweden2017In: Resources, Conservation and Recycling, ISSN 0921-3449, E-ISSN 1879-0658, Vol. 125, p. 86-97Article in journal (Refereed)
    Abstract [en]

    The option of producing the chemical succinic acid from bio-based resources is well in line with current political and industrial ambitions for a bio-based economy. A little explored but intriguing biomass feedstock opportunity is food waste. Mixed food waste is especially appealing as it represents less resource competition than more homogenous food waste fractions. The feasibility of producing succinic acid from mixed food waste depends on both technical and societal system structures. Therefore, to assess the production prospect, it is important to investigate all relevant system components. This study explores from such multiple perspectives the feasibility of chemical production as a viable added pathway for mixed food waste, using microbial production of succinic acid from municipal solid waste in Sweden as an example. The perspectives explored are: 1) feedstock feasibility, 2) societal drivers and barriers for technology progress, and 3) resource availability. Findings show that even though, from a technical feasibility and resource availability perspective, production seems possible, it lacks institutional support and actor commitment and alignment for development in Sweden. Findings also show that a holistic and interdisciplinary systems perspective contributes valuable insight when assessing prospects for bio-based chemicals.

  • 14. 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.

  • 15.
    Råvik, Mattias
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Barnfield Frej, Kine
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Effect of bacterial cell surface properties on membrane filtration and chromatography media interactionManuscript (Other academic)
  • 16.
    Råvik, Mattias
    et al.
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    Cimander, Christian
    Elofsson, Ulla
    Veide, Andres
    KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT.
    A method for microbial cell surface fingerprinting based on surface plasmon resonance2007In: Journal of Biochemical and Biophysical Methods, ISSN 0165-022X, E-ISSN 1872-857X, Vol. 70, no 4, p. 595-604Article in journal (Refereed)
    Abstract [en]

    A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).

  • 17.
    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)
  • 18. Shokri, Atefeh
    et al.
    Veide, Andres
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    RelA1 gene control of Escherichia coli lipid structure and cell performace during glucose limited fed-batch conditions2006In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 73, no 2, p. 464-473Article in journal (Refereed)
    Abstract [en]

    At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.

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