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  • 1. Castan, A.
    et al.
    Nasman, A.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone2002Inngår i: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 30, nr 7, s. 847-854Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 2.
    Ibarra, David
    et al.
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi.
    Köpcke, Viviana
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi.
    Ek, Monica
    KTH, Skolan för kemivetenskap (CHE), Fiber- och polymerteknik, Träkemi och massateknologi.
    Behavior of different monocomponent endoglucanases on the accessibility and reactivity of dissolving-grade pulps for viscose process2010Inngår i: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 47, s. 355-362Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Three different commercial monocomponent endoglucanases, with and without a cellulose-binding domain (CBD) and differences in their glycosidic hydrolysis mechanisms, were compared with respect to their ability to enhance the accessibility and reactivity of dissolving-grade pulps for viscose production. Hardwood (eucalyptus) and softwood (mixture of Norway spruce and Scots pine) commercial dried and never-dried bleached sulfite dissolving pulps were used for this purpose. The effects of the enzymatic treatments on pulps were studied by reactivity, according to Fock's method, and viscosity measurements, and recording of molecular weight distributions. Among the different assayed enzymes, endoglucanase with a CBD and an inverting hydrolysis mechanism was found to be the most effective in increasing the reactivity of both pulps. Simultaneously, the viscosity decreased, being more marked for softwood dissolving pulp. A narrower molecular weight distribution, with a great reduction in the amount of long-chain cellulose molecules was observed in both pulps, being more pronounced for softwood dissolving pulp. By contrast, endoglucanase without a CBD and a retaining hydrolysis mechanism showed a barley enhancement of the studied properties. The effects of the different endoglucanase treatments were more pronounced when never-dried dissolving pulps were used.

  • 3. Ignatova, Z.
    et al.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner                               , Bioteknologi.
    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 coli2000Inngår i: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 26, nr 04-feb, s. 165-170Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 4. Rozkov, A.
    et al.
    Schweder, T.
    Veide, Andres
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Enfors, Sven-Olof
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins2000Inngår i: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 27, nr 10, s. 743-748Artikkel i tidsskrift (Fagfellevurdert)
    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.

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