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Sökning: L773:1873 4863 OR L773:0168 1656

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1.
  • Nilsson, Anneli, et al. (författare)
  • Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol
  • 2001
  • Ingår i: Journal of Biotechnology. - 1873-4863 .- 0168-1656. ; 89:1, s. 41-53
  • Tidskriftsartikel (refereegranskat)abstract
    • Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(t.max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations. (C) 2001 Elsevier Science B.V. All rights reserved.
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2.
  • Bachinger, T., et al. (författare)
  • Monitoring cellular state transitions in a production-scale CHO-cell process using an electronic nose
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:1, s. 61-71
  • Tidskriftsartikel (refereegranskat)abstract
    • An electronic nose is used to monitor the bioreactor off-gas composition in perfused cultivations of a CHO-cell line producing recombinant human blood coagulation factor VIII. The applicability of the electronic nose for monitoring cellular state transitions and process control is explained. It is shown that the instrument can reveal characteristic process states related to product and lactate formation, and detect microbial infections in a very early stage of the infection. The visualization of ideal process conditions is realized by using principal component analysis (PCA) and the on-line applicability of this method is outlined. The results illustrate the potential of the electronic nose as on-line sensor for ensuring product and process quality in production-scale bioprocesses. Copyright (C) 2000 Elsevier Science B.V.An electronic nose is used to monitor the bioreactor off-gas composition in perfused cultivations of a CHO-cell line producing recombinant human blood coagulation factor VIII. The applicability of the electronic nose for monitoring cellular state transitions and process control is explained. It is shown that the instrument can reveal characteristic process states related to product and lactate formation, and detect microbial infections in a very early stage of the infection. The visualization of ideal process conditions is realized by using principal component analysis (PCA) and the on-line applicability of this method is outlined. The results illustrate the potential of the electronic nose as on-line sensor for ensuring product and process quality in production-scale bioprocesses.
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3.
  • Bandmann, Nina, et al. (författare)
  • Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins
  • 2002
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 93:1, s. 1-14
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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4.
  • Bandmann, Nina, et al. (författare)
  • Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 79:2, s. 161-172
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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5.
  • Christakopoulos, Paul, et al. (författare)
  • Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3
  • 1996
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 51:2, s. 181-189
  • Tidskriftsartikel (refereegranskat)abstract
    • Two low molecular mass endo-1,4-β-d-xylanases from Fusarium oxysporum were purified to homogeneity by gel-filtration and ion-exchange chromatography. They exhibit molecular masses of 20.8 (xylanase I) and 23.5 (xylanase II) kDa, and isoelectric points of 9.5 and 8.45–8.70, respectively. Both xylanases display remarkable pH (9.0) stability. At 40 to 55 °C xylanase II is more thermostable than xylanase I but less active on xylan. In contrast to xylanase I, xylanase II is able to hydrolyze 1-O-4-methylumbelliferyl-(β-d-glucopyranosyl)-β-d-xylopyranoside (muxg). Neither of these enzymes hydrolyze xylotriose. They bind on crystalline cellulose but not on insoluble xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that both enzymes cleave preferentially the internal glycosidic bonds of xylopentaose and oat spelts xylan. Thus the purified enzymes appeared to be true endo-β-1,4-xylanases. The amino terminal sequences of xylanases I and II show no homology. Xylanase I shows high similarity with alkaline low molecular mass xylanases of family G/11.
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6.
  • Christakopoulos, Paul, et al. (författare)
  • Purification and mode of action of a low molecular mass endo-1,4-β-d-glucanase from Fusarium oxysporum
  • 1995
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 39:1, s. 85-93
  • Tidskriftsartikel (refereegranskat)abstract
    • A low molecular mass (23.2 kDa) endo-1,4-β-d-glucanase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme was optimally active at pH 6.0 and at 50 ° C. It had a pI value of 8.6 and was stable at 55 ° C for 1 h. It hydrolyzed carboxymethylcellulose, cello-oligosaccharides (Glcn) and 4-methylumbelliferylcello-oligosaccharides but did not hydrolyze cellobiose, p-nitrophenyl β-o-glucoside, p-nitrophenyl β-d-xyloside, Avicel, filter paper and xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that this enzyme cleaved preferentially the internal glycoside bonds of higher cello-oligosaccharides. The enzyme also catalyzed the formation of transfer products in the presence of cellotriose, cellotetraose and 4-methylumbelliferylglucoside (MeUmbGlc).
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7.
  • Christov, L, et al. (författare)
  • Effects of purified endo-β-1,4-xylanases of family 10 and 11 and acetyl xylan esterases on eucalypt sulfite dissolving pulp
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 83:3, s. 231-244
  • Tidskriftsartikel (refereegranskat)abstract
    • Sulfite dissolving pulp from Eucalyptus grandis contained approximately 3.8% O-acetyl-4-O-methylglucuronoxylan with a molar ratio of xylose:4-O-methylglucuronic acid:acetyl group close to 13.6:1:6.2. The effects produced by purified endo-xylanases from two different glycosyl hydrolase families (family 10 and 11) as well as acetyl xylan esterases were examined and assessed on pulp in relation to their bleaching abilities. The purified endo-xylanases hydrolyzed only a limited portion (less than 30%) of the acetylglucuronoxylan present in the pulp. The enzymes of family 10 produced acetylated xylobiose and xylotriose whereas acetylated xylobiose was not observed among the products released from the pulp by the family 11 xylanases. The esterases however were not capable of deacetylating the acetylated aldouronic acids generated by the xylanases. Regardless of the different mode of action of the endo-xylanases on dissolving pulp, their effect on pulp bleaching was not related to the amount and nature of sugars generated or the glycosyl hydrolase family. No additional brightness gain was obtained when endo-xylanases were used in conjunction with acetyl xylan esterases, suggesting that the latter do not play an important role in biobleaching of eucalypt sulfite dissolving pulps. Copyright (C) 2000 Elsevier Science B.V. Sulfite dissolving pulp from Eucalyptus grandis contained approximately 3.8% O-acetyl-4-O-methylglucuronoxylan with a molar ratio of xylose:4-O-methylglucuronic acid:acetyl group close to 13.6:1:6.2. The effects produced by purified endo-xylanases from two different glycosyl hydrolase families (family 10 and 11) as well as acetyl xylan esterases were examined and assessed on pulp in relation to their bleaching abilities. The purified endo-xylanases hydrolyzed only a limited portion (less than 30%) of the acetylglucuronoxylan present in the pulp. The enzymes of family 10 produced acetylated xylobiose and xylotriose whereas acetylated xylobiose was not observed among the products released from the pulp by the family 11 xylanases. The esterases however were not capable of deacetylating the acetylated aldouronic acids generated by the xylanases. Regardless of the different mode of action of the endo-xylanases on dissolving pulp, their effect on pulp bleaching was not related to the amount and nature of sugars generated or the glycosyl hydrolase family. No additional brightness gain was obtained when endo-xylanases were used in conjunction with acetyl xylan esterases, suggesting that the latter do not play an important role in biobleaching of eucalypt sulfite dissolving pulps.
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8.
  • Cimander, C., et al. (författare)
  • Integration of distributed multi-analyzer monitoring and control in bioprocessing based on a real-time expert system
  • 2003
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 103:3, s. 237-248
  • Tidskriftsartikel (refereegranskat)abstract
    • A computer system solution for integration of a distributed bioreactor monitoring and control instrumentation on the laboratory scale is described. Bioreactors equipped with on-line analyzers for mass spectrometry, near-infrared spectroscopy, electrochemical probes and multi-array gas sensors and their respective software were networked through a real-time expert systems platform. The system allowed data transmission of more than 1800 different signals from the instrumentation, including signals from gas sensors, electrodes, spectrometer detectors, balances, flowmeters, etc., and were used for processing and carrying out a number of computational tasks such as partial least-square regression, principal component analysis, artificial neural network modelling, heuristic decision-making and adaptive control. The system was demonstrated on different cultivations/fermentations which illustrated sensor fusion control, multivariate statistical process monitoring, adaptive glucose control and adaptive multivariate control. The performance of these examples showed high operational stability and reliable function and meet typical requirements for production safety and quality. © 2003 Elsevier B.V. All rights reserved.
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9.
  • Cimander, C., et al. (författare)
  • Sensor fusion for on-line monitoring of yoghurt fermentation
  • 2002
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 99:3, s. 237-248
  • Tidskriftsartikel (refereegranskat)abstract
    • Measurement data from an electronic nose (EN), a near-infrared spectrometer (NIRS) and standard bioreactor probes were used to follow the course of lab-scale yoghurt fermentation. The sensor signals were fused using a cascade neural network: a primary network predicted quantitative process variables, including lactose, galactose and lactate, a secondary network predicted a qualitative process state variable describing critical process phases, such as the onset of coagulation or the harvest time. Although the accuracy of the neural network prediction was acceptable and comparable with the off-line reference assay, its stability and performance were significantly improved by correction of faulty data. The results demonstrate that on-line sensor fusion with the chosen analyzers improves monitoring and quality control of yoghurt fermentation with implications to other fermentation processes. ⌐ 2002 Elsevier Science B.V. All rights reserved.
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10.
  • Doverskog, M., et al. (författare)
  • Determination of NADH-dependent glutamate synthase (GOGAT) in Spodoptera frugiperda (Sf9) insect cells by a selective H-1/N-15 NMR in vitro assay
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 79:1, s. 87-97
  • Tidskriftsartikel (refereegranskat)abstract
    • This is the second of two papers [Drews, M., Doverskog, M., Qhman, L., Chapman, B.E., Jacobsson, U., Kuchel, P.W., Haggstrom, L., 2000. Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR. J. Biotechnol. 78, 23-37]. where the general goal has been to determine and characterise the glutamine metabolism in Sf9 cells. The presence of glutamate synthase (GOGAT) activity was investigated in cell-free extracts of S. frugiperda (Sf9) insect cells by modified H-1/N-15 spin-echo and gradient enhanced multiple quantum coherence NMR spectroscopy techniques. Cell-free extracts were prepared from cells cultured in a serum-free medium. The assay conditions were based on conventional spectrophotometric and chromatographic methods. NMR data showed that nitrogen from [5-N-15] glutamine was selectively incorporated into 2-oxoglutarate forming [2-N-15] glutamate with a specific activity of 4.15 +/- 0.21 nmol [2-N-15] glutamate min (-1) (mg total protein)(-1) in the cell-free extracts. The enzyme activity was exclusively dependent on NADH as coenzyme and was completely inhibited by 1 mM azaserine. From the results obtained, we conclude that Sf9 cells possess NADH-GOGAT activity. Furthermore, the high specificity of the NMR method enables distinction of competing reactions from glutaminase and glutamate dehydrogenase.
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11.
  • Drews, M., et al. (författare)
  • Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells : evidence for the presence of the nitrogen assimilation system, and a metabolic switch by H-1/N-15 NMR
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 78:1, s. 23-37
  • Tidskriftsartikel (refereegranskat)abstract
    • H-1/N-15 and C-13 NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-N-15]glutamine, [5-N-15]glutamine, [2-N-15]glutamate, (NH4Cl)-N-15, [2-N-15]alanine, and [1-C-13]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. H-1/N-15 NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media (NH4+)-N-15 was consumed and incorporated into alanine. (NH4+)-N-15 was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. C-13 NMR revealed that the [1-C-13] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.
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12.
  • Enfors, Sven-Olof, et al. (författare)
  • Physiological responses to mixing in large scale bioreactors
  • 2001
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 85:2, s. 175-185
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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13.
  • Greiner, R., et al. (författare)
  • Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 84:1, s. 53-62
  • Tidskriftsartikel (refereegranskat)abstract
    • Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate- degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P5, D/L-Ins(2,3,4,5)P4, D/L-Ins(2,4,5)P3 or D/L-Ins(1,2,4)P3, D/L-Ins(1,2)P2 or Ins(2,5)P2 or D/L-Ins(4,5)P2 to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo- inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D- Ins(1,2,3,5,6)P5 isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F1 and F2 from wheat bran and the myo- inositol phosphates produced by fraction F2. Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P5, D- Ins(2,3,4,5)P4, D-Ins(2,4,5)P3, Ins(2,5)P2 to finally Ins(2)P (notation 6/1/3/4/5). (C) 2000 Elsevier Science B.V.Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P5, D/L-Ins(2,3,4,5)P4, D/L-Ins(2,4,5)P3 or D/L-Ins(1,2,4)P3, D/L-Ins(1,2)P2 or Ins(2,5)P2 or D/L-Ins(4,5)P2 to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1,2,3,5,6)P5 isomer. The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P5, D-Ins(2,3,4,5)P4, D-Ins(2,4,5)P3, Ins(2,5)P2 to finally Ins(2)P (notation 6/1/3/4/5).
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14.
  • Gräslund, Susanne, et al. (författare)
  • A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
  • 2002
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 99:1, s. 41-50
  • Tidskriftsartikel (refereegranskat)abstract
    • An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.
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15.
  • Gräslund, Torbjörn, et al. (författare)
  • Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology
  • 2002
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 96:1, s. 93-102
  • Tidskriftsartikel (refereegranskat)abstract
    • The highly charged domain Z(basic) can be used as a fusion partner to enhance adsorption of target proteins to cation exchanging resins at high pH-values. In this paper, we describe a strategy for purification of target proteins fused to Z(basic) at a constant physiological pH using cation exchange chromatography in an expanded bed mode. We show that two proteins, Klenow DNA polymerase and the viral protease 3C, can be efficiently purified from unclarified Escherichia coli homogenates in a single step with a selectivity analogous to what is normally achieved by affinity chromatography. The strategy also includes an integrated site-specific removal of the Z(basic) purification handle to yield a free target protein.
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16.
  • Gulich, S., et al. (författare)
  • Protein engineering of an IgG-binding domain allows milder elution conditions during affinity chromatography
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:03-feb, s. 233-244
  • Tidskriftsartikel (refereegranskat)abstract
    • One of the problems in the recovery of antibodies by affinity chromatography is the low pH, which is normally essential to elute the bound material from the column. Here, we have addressed this problem by constructing destabilized mutants of a domain analogue (domain Z) from an IgG-binding bacterial receptor, protein A. In ol-der to destabilize the IgG-binding domain, two protein engineered variants were constructed using site-directed mutagenesis of the second loop of this antiparallel three-helix bundle domain. In the first mutant (Z6C), the second loop was extended with six glycines in order to evaluate the significance of the loop length. In the second mutant (ZL4G), the original loop sequence was exchanged for glycines in order to evaluate the importance of the loop forming residues. Both mutated variants have a lower a-helical content, as well as a lower thermal and chemical stability compared to the parent 2-molecule. The affinity to IgG was slightly lowered in both cases, mainly due to higher dissociation rates. Interestingly, the elution studies showed that most of the bound IgG-molecules could be eluted at a pH as high as 4.5 from columns with the engineered ligands, while only 70% of the bound IgG could be eluted from the matrix with the parent Z as ligand.
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17.
  • Gulich, S., et al. (författare)
  • Stability towards alkaline conditions can be engineered into a protein ligand
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 169-178
  • Tidskriftsartikel (refereegranskat)abstract
    • One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting of replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.
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18.
  • Han, L., et al. (författare)
  • 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 spectroscopy
  • 2002
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 92:3, s. 237-249
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
19.
  • Henriksson, Gunnar, et al. (författare)
  • A critical review of cellobiose dehydrogenases
  • 2000
  • Ingår i: Journal of Biotechnology. - : ELSEVIER SCIENCE BV. - 0168-1656 .- 1873-4863. ; 78:2, s. 93-113
  • Forskningsöversikt (refereegranskat)abstract
    • Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron accepters including quinones, phenoxyradicals, Fe3+, Cu2+ and tiiodide ion. Monosaccharides, maltose and molecular oxygen are:poop substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron accepters, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.
  •  
20.
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21.
  • Jahic, Mehmedalija, et al. (författare)
  • Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
  • 2003
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 102:1, s. 45-53
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
22.
  • Jonasson, P., et al. (författare)
  • Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:03-feb, s. 215-226
  • Tidskriftsartikel (refereegranskat)abstract
    • An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.
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23.
  • Kalogeris, E., et al. (författare)
  • Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus : Characterization of two isozymes
  • 1998
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 60:3, s. 155-163
  • Tidskriftsartikel (refereegranskat)abstract
    • Production of xylanases by the thermophilic fungus Thermoascus aurantiacus under solid state culture (SSC) was enhanced by optimization of the type of carbon and nitrogen source, inoculum type, moisture level and particle size of the carbon source. Under these conditions, yields as high as 6193 U g−1 of carbon source were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl-β-glycosides of xylose (MUX) and xylobiose (MUX2) were used to characterize xylanase multienzyme components, after separation by isoelectric focusing. The zymogram indicated one major and two minor xylanases and one β-xylosidase. The major (xylanase I) and one of the minor (xylanase II) xylanases were separated and characterized. Both xylanases exhibited remarkable thermostability.
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24.
  • Kepka, C., et al. (författare)
  • Pilot-scale extraction of an intracellular recombinant cutinase from E-coli cell homogenate using a thermoseparating aqueous two-phase system
  • 2003
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 103:2, s. 165-181
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
25.
  • Larsson, M., et al. (författare)
  • High-throughput protein expression of cDNA products as a tool in functional genomics
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 143-157
  • Tidskriftsartikel (refereegranskat)abstract
    • A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.
  •  
26.
  • Nord, K., et al. (författare)
  • Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1(M) and Taq DNA polymerase
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:1, s. 45-54
  • Tidskriftsartikel (refereegranskat)abstract
    • Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Tag DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0.5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.
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27.
  • Samuelson, Patrik, et al. (författare)
  • Display of proteins on bacteria
  • 2002
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 96:2, s. 129-154
  • Forskningsöversikt (refereegranskat)abstract
    • Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and disc-asses current uses and possible future trends of these emerging technologies.
  •  
28.
  • Sterky, Fredrik, et al. (författare)
  • Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR
  • 1998
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 60:1-2, s. 119-129
  • Tidskriftsartikel (refereegranskat)abstract
    • Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads. An arbitrary primer initiates extension from the unknown region and back towards the known locus. The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification. The PCR products are then directly sequenced by solid-phase or cycle sequencing. The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.
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29.
  • Sterky, Fredrik, et al. (författare)
  • Sequence analysis of genes and genomes
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:1, s. 1-31
  • Forskningsöversikt (refereegranskat)abstract
    • A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.
  •  
30.
  • Strandberg, L, et al. (författare)
  • Expression and characterization of a tripartite fusion protein consisting of chimeric IgG-binding receptors and beta-galactosidase
  • 1990
  • Ingår i: Journal of Biotechnology. - : Elsevier. - 0168-1656 .- 1873-4863. ; 13:1, s. 83-96
  • Tidskriftsartikel (refereegranskat)abstract
    • Using protein engineering, a tripartite fusion protein was constructed consisting of five IgG-binding regions of protein A from Staphylococcus aureus, two IgG-binding regions of protein G from Streptococcus strain G148 and beta-galactosidase from Escherichia coli. The resulting protein lacks the serum albumin binding regions of native protein G. The fusion protein, which is a tetramer of approximately 660 kDa, was designed as a tool for immunological assays taking advantage of its broad spectrum of antibody affinity. The gene was placed under control of two promoters, the PR promoter and the lac UV5 promoter and the expression from the two promoters was studied in a bioreactor. Induction of the PR promoter gave an intracellular product concentration corresponding to 20% of the cell dry weight. By utilizing the properties of beta-galactosidase, the protein was purified by extraction in an aqueous two-phase system. The fusion protein was not proteolytically degraded during the cultivation and purification steps. The biological activity of all three parts of the protein was demonstrated with a competitive ELISA.
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31.
  • Topakas, E., et al. (författare)
  • Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water–organic solvent mixtures
  • 2003
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 102:1, s. 33-44
  • Tidskriftsartikel (refereegranskat)abstract
    • An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography. The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively. The enzyme was optimally active at pH 7 and 45 °C. The purified esterase was fully stable at pH 7.0–9.0 and temperature up to 45 °C after 1 h incubation. Determination of kcat/Km revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-α-l-arabinofuranoside. In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S. thermophile xylanase. FAE-II by itself could release only little ferulic acid from destarched wheat bran. The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water.
  •  
32.
  • Vogel, H J, et al. (författare)
  • An In Vivo 31P NMR Comparison of Freely Suspended and Immobilized Catharanthus roseus Plant Cells
  • 1984
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 1, s. 159-170
  • Tidskriftsartikel (refereegranskat)abstract
    • 31P NMR spectra obtained for Catharanthus roseus plant cells entrapped in agarose or alginate were compared to those obtained for freely suspended cells. Oxygenated buffer was circulated through the NMR tube in all three instances. Essentially no differences were observed in the levels of the major metabolites: ATP, NAD(H), UDPG, cytoplasmic Pi, and sugar phosphates. Furthermore, the saturation of ATP by Mg2+, the energy status as determined from the ADP/ATP ratio, as well as the intracellular cytoplasmic and vacuolar pH, were not affected by the entrapment in the polymers. Since the cytoplasmic pH for freely suspended cells drops about 0.5 pH units upon shifting from aerobic to anaerobic conditions, these results indicate that the majority of the entrapped cells are properly oxygenated and that products such as CO2 can diffuse away. In addition, we found that the uptake rate of Pifrom the medium was lower in the polymer entrapped cells, and that the utilization rate of Pi from the vacuolar pool was also considerably reduced. However, the same pattern of Pi uptake, storage and utilization was observed in all cases. Thus, all the results obtained with the noninvasive 31P NMR technique suggest that the entrapment in agarose or alginate does not adversely affect cell metabolism since the phosphate metabolism and the cytoplasmic pH appear unaltered. 
  •  
33.
  • Wernérus, Henrik, et al. (författare)
  • Engineering of staphylococcal surfaces for biotechnological applications
  • 2002
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 96:1, s. 67-78
  • Forskningsöversikt (refereegranskat)abstract
    • Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.
  •  
34.
  • Zhang, Jing, et al. (författare)
  • Polygalacturonase is the key component in enzymatic retting of flax
  • 2000
  • Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 81:1, s. 85-89
  • Tidskriftsartikel (refereegranskat)abstract
    • Seven commercial enzyme mixtures were tested for their ability to perform retting of flax (i.e. to separate flax fibers by partly removal of middle lamella) and were assayed for hydrolysis of xylan, cellulose and four kinds of pectin. The only activity that showed correlation to the ability to perform retting was the degradation of low esterfied pectin. A purified Aspergillus niger polygalacturonase was also shown to be able to perform retting. From this data it is hypothesized that degradation of the smooth regions (i.e. non-methylated polygalacturonase) in the middle lamella pectin is the most important step in enzymatic retting.
  •  
35.
  • Ahlqvist, Josefin, et al. (författare)
  • Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies
  • 2006
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 122:2, s. 216-225
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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36.
  • Andersson, Henrik, et al. (författare)
  • Assaying cardiac biomarkers for toxicity testing using biosensing and cardiomyocytes derived from human embryonic stem cells
  • 2010
  • Ingår i: JOURNAL OF BIOTECHNOLOGY. - : Elsevier Science B.V., Amsterdam.. - 0168-1656 .- 1873-4863. ; 150:1, s. 175-181
  • Tidskriftsartikel (refereegranskat)abstract
    • Human embryonic stem cell (hESC) derived cardiomyocytes are in the present study being used for testing drug-induced cardiotoxicity in a biosensor set-up. The design of an in vitro testing alternative provides a novel opportunity to surpass previous methods based on rodent cells or cell lines due to its significantly higher toxicological relevance. In this report we demonstrate how hESC-derived cardiomyocytes release detectable levels of two clinically decisive cardiac biomarkers, cardiac troponin T and fatty acid binding protein 3, when the cardiac cells are exposed to the well-known cardioactive drug compound. doxorubicin. The release is monitored by the immuno-biosensor technique surface plasmon resonance, particularly appropriate due to its capacity for parallel and high-throughput analysis in complex media.
  •  
37.
  • Andersson, Louise, et al. (författare)
  • Draft genome sequence of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB strain BBA69670
  • 2016
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 222, s. 11-12
  • Tidskriftsartikel (refereegranskat)abstract
    • Rhizoctonia solani is a widespread plant pathogenic fungus featuring a broad host range including several economically important crops. Accordingly, genome analyses of R. solani isolates are important to uncover their pathogenic potential. Draft genome sequences for four R. solani isolates representing three of the 14 R. solani anastomosis groups (AGs) are available. Here, we present the first draft genome sequence for an R. solani AG2-2IIIB isolate that is pathogenic on sugar beet. The fungal genome was assembled in 2065 scaffolds consisting of 5826 contigs amounting to a size of about 52 Mb which is larger than any other R. solani isolate known today. Genes potentially encoding cellulolytic, lignolytic and pectinolytic enzymes were identified. (c) 2016 Elsevier B.V. All rights reserved.
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38.
  • Andersson, Mariette, et al. (författare)
  • Targeted gene suppression by RNA interference: An efficient method for production of high-amylose potato lines
  • 2006
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 123:2, s. 137-148
  • Tidskriftsartikel (refereegranskat)abstract
    • Production of high-amylose potato lines can be achieved by inhibition of two genes coding for starch branching enzymes. The use of antisense technology for gene inhibition have yielded a low frequency of high-amylose lines that mostly was correlated with high numbers of integrated T-DNA copies. To investigate whether the production of high-amylose lines could be improved, RNA interference was used for gene inhibition of the genes Sbe1 and Sbe2. Two constructs with 100 bp segments (pHAS2) or 200 bp segments (pHAS3) of both branching enzyme genes were cloned as inverted repeats controlled by a potato granule-bound starch synthase promoter. The construct pHAS3 was shown to be very efficient, yielding high-amylose quality in more than 50% of the transgenic lines. An antisense construct, included in the study as a comparator, resulted in only 3% of the transgenic lines being of high-amylose type. Noticeable was also that pHAS3 yielded low T-DNA copy inserts with an average of 83% of backbone-free transgenic lines being single copy events.
  •  
39.
  • Andrade-Pavón, Dulce, et al. (författare)
  • Inhibition of recombinant enzyme 3-hydroxy-3-methylglutaryl-CoA reductase from Candida glabrata by α-asarone-based synthetic compounds as antifungal agents
  • 2019
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 292, s. 64-67
  • Tidskriftsartikel (refereegranskat)abstract
    • Due to increasing resistance of Candida species to antifungal drugs, especially azoles, new drugs are needed. The proposed compounds 3 and 4 are analogous to α-asarone (2), a naturally occurring potent inhibitor of HMGR with hypolipidemic and antifungal activity. We used the recombinant enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Candida glabrata (CgHMGR) as a model to test the effectiveness of the test compounds. Compounds 3 and 4 demonstrated inhibitory kinetics, having lower IC 50 values (42.65 μM and 28.77 μM, respectively) than compound 2 (>100 μM). The docking studies showed better binding energies for compounds 3 and 4 (−5.35 and −6.1 kcal/mol, respectively) than for compound 2 (−4.53 kcal/mol). These findings suggest that the tested compounds are better than their natural analogue. Plaque assays were performed on the C. glabrata strain CBS138 by applying ergosterol or cholesterol to evaluate the possible reversal of the inhibition induced by compounds 2, 3 and 4. Inhibition was easily suppressed in all three cases, recovering the viability of C. glabrata. These results reveal that the CgHMGR model is excellent for testing antifungals. Compound 4 produced the best effect and is herein proposed as a new potent antifungal agent.
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40.
  • Bettiga, Maurizio, et al. (författare)
  • Acoustophoretic Separation of Yeast Cells
  • 2010
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 150:Suppl. 1, s. 522-522
  • Konferensbidrag (refereegranskat)
  •  
41.
  •  
42.
  • Borg, Jörgen, et al. (författare)
  • Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.
  • 2004
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 114:1-2, s. 21-30
  • Tidskriftsartikel (refereegranskat)abstract
    • Methods currently used for surface display on insect cells and budded baculovirus, all utilize the sequences from class I transmembrane proteins. This gives rise to some problems when handling unknown genes or cDNAs encoding full-length proteins. First, the stop codon from the cloned gene will be located upstream of the sequence for the transmembrane region. Second, the chance of getting the sequences encoding the signal peptide and the transmembrane region in frame with the cloned gene is small. To minimize these problems, we here present a method by which cDNAs or genes of interest can be cloned and fused to the codons for the signal peptide and transmembrane region of neuraminidase (NA), a class II transmembrane protein of the influenza virus. By placing both the signal peptide and transmembrane region at the amino-terminal, potential problems regarding stop codons are eliminated and errors in frame-shift minimized. To obtain proof of principle, the gene encoding enhanced green fluorescent protein, EGFP, was subcloned into a shuttle vector downstream of the neuraminidase sequence and the fusion product was then transferred to a baculovirus vector and transfected into insect cells (Sf9). Using this method, EGFP was found to be expressed on the surface of both infected cells and budded virus in an accessible manner.
  •  
43.
  • Bradoo, Sapna, et al. (författare)
  • Synthesis of alkylgalactosides using whole cells of Bacillus pseudofirmus species as catalysts
  • 2004
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 110:3, s. 273-285
  • Tidskriftsartikel (refereegranskat)abstract
    • Whole cells of alkaliphilic Bacillus pseudofirmus AR-199, induced for beta-galactosidase activity, were used for the synthesis of 1-hexyl-beta-D-galactoside and 1-octyl-beta-D-galactoside, respectively, by transglycosylation reaction between lactose and the corresponding alcohol acceptor. The product yield was strongly influenced by the initial water content in the reaction mixture. Water content of 10% (v/v) was optimal providing 3.6-36 mM hexyl galactoside from 10 to 150 mM lactose, and no secondary product hydrolysis. Product yield could be enhanced by supplementing the reaction mixture with more cells or partly replacing the product with fresh substrate, but was decreased with time to the initial equilibrium level. Cell permeabilisation or disruption resulted in increased reaction rate and higher product yield but was followed by product hydrolysis. Octyl galactoside synthesis using whole cells was optimal at water content of 2% (v/v) with a yield of 26%. The cells were immobilised in cryogels of polyvinyl alcohol for use in continuous process, where hexyl galactoside was produced with a constant yield of 50% from 50 mM lactose for at least a week. (C) 2004 Elsevier B.V. All rights reserved.
  •  
44.
  • Brolen, Gabriella, et al. (författare)
  • Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage
  • 2010
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 145:3, s. 284-294
  • Tidskriftsartikel (refereegranskat)abstract
    • Human embryonic stem cells offer a potential unlimited supply for functional hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing various hepatic markers. These cells could be used in various applications such as studies of drug metabolism and hepatotoxicity, which however, would require a significant expression of drug metabolizing enzymes. To derive these cells we use a stepwise differentiation protocol where growth- and maturation factors are added. The first phase involves the formation of definitive endoderm. Next, these cells are treated with factors known to promote the induction and proliferation towards hepatic progenitor cell types. In the last phase the cells are terminally differentiated and maturated into functional hepatocyte-like cells. The cultures were characterized by analysis of endodermal or hepatic markers and compared to cultures derived without induction via definitive endoderm. Hepatic functions such as urea secretion, glycogen storage, indocyanine green uptake and secretion, and cytochrome P450-expression and activity were evaluated. The DE-Hep showed a hepatocyte morphology with sub-organized cells and exhibited many liver-functions including transporter activity and capacity to metabolize drugs specific for important cytochrome P450 sub-families. This represents an importantstep in differentiation of hESC into functional hepatocytes. (C) 2009 Elsevier B.V. All rights reserved.
  •  
45.
  • Buehligen, Franziska, et al. (författare)
  • Analysis of aging in lager brewing yeast during serial repitching
  • 2014
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 187, s. 60-70
  • Tidskriftsartikel (refereegranskat)abstract
    • Serial repitching of brewing yeast inoculates is an important economic factor in the brewing industry, as their propagation is time and resource intensive. Here, we investigated whether replicative aging and/or the population distribution status changed during serial repitching in three different breweries with the same brewing yeast strain but different abiotic backgrounds and repitching regimes with varying numbers of reuses. Next to bud scar numbers the DNA content of the Saccharomyces pastorianus HEBRU cells was analyzed. Gene expression patterns were investigated using low-density microarrays with genes for aging, stress, storage compound metabolism and cell cycle. Two breweries showed a stable rejuvenation rate during serial repitching. In a third brewery the fraction of virgin cells varied, which could be explained with differing wort aeration rates. Furthermore, the number of bud scars per cell and cell size correlated in all 3 breweries throughout all runs. Transcriptome analyses revealed that from the 6th run on, mainly for the cells positive gene expression could be seen, for example up-regulation of trehalose and glycogen metabolism genes. Additionally, the cells' settling in the cone was dependent on cell size, with the lowest and the uppermost cone layers showing the highest amount of dead cells. In general, cells do not progressively age during extended serial repitching.
  •  
46.
  • Buehligen, Franziska, et al. (författare)
  • Sustainability of industrial yeast serial repitching practice studied by gene expression and correlation analysis
  • 2013
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 168:4, s. 718-728
  • Tidskriftsartikel (refereegranskat)abstract
    • Bottom-fermenting Saccharomyces pastorianus strains driving brewing fermentation processes are usually reused several times. It is still unclear, whether the number of successions may have an impact on cell physiology prompting consequences for brewing quality. In this study, fermentation performance of up to twenty consecutive runs in a brewery was investigated. For each run mRNA expression levels of cellular marker molecules, which are known to correlate with metabolism, hexose transport, aging processes, stress response mechanisms and flocculation capability was estimated to obtain information on changes in cell physiology over the successive runs. Low-density microarrays were used for this purpose and the resulting gene expression profiles were finally correlated with changes in the abiotic micro-environments. A surprising stability of the marker molecule expression profiles within each specific serial repitching was stated. Loss of flocculation or an advanced aging could not be detected during serial repitching in the analyzed brewery. However, certain runs of the serial repitchings showed high variation in stress response which was found to be caused by perturbations of the abiotic conditions. Regardless, the study showed that S. pastorianus can be used repeatedly in serial repitching processes without loss of prominent physiological characteristics.
  •  
47.
  • Bäcklund, Emma, et al. (författare)
  • Fedbatch design for periplasmic product retention in Escherichia coli
  • 2008
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 135:4, s. 358-365
  • Tidskriftsartikel (refereegranskat)abstract
    • The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h-1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.
  •  
48.
  • Cassland, Pierre, et al. (författare)
  • Heterologous expression of barley and wheat oxalate oxidase in an E-coli trxB gor double mutant
  • 2004
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 109:1-2, s. 53-62
  • Tidskriftsartikel (refereegranskat)abstract
    • Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.
  •  
49.
  • Charoenrat, Theppanya, et al. (författare)
  • Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth
  • 2006
  • Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 122:1, s. 86-98
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
  •  
50.
  • Chen, Shan, et al. (författare)
  • Characterization of the stability of Vibrio fluvialis JS17 amine transaminase
  • 2018
  • Ingår i: Journal of Biotechnology. - : Elsevier. - 0168-1656 .- 1873-4863. ; 282, s. 10-17
  • Tidskriftsartikel (refereegranskat)abstract
    • The amine transaminase from Vibrio fluvialis (Vf-ATA) is an attractive enzyme with applications within Biocatalysis for the preparation of chiral amines. Various catalytic properties of Vf-ATA have been investigated, but a biophysical characterization of its stability has been lacking. Today, the industrial application of Vf-ATA is limited by its low operational stability. In order to enhance the knowledge regarding the structural stability of ATAs, general characterizations of different ATAs are required. In this work, the stability of Vf-ATA was explored. First, the affinity between enzyme and pyridoxal-5’-phosphate (PLP) (KD value of 7.9 ΌM) was determined. Addition of PLP to enzyme preparations significantly improved the enzyme thermal stability by preventing enzyme unfolding. With the aim to understand if this was due to the PLP phosphate group coordination into the phosphate group binding cup, the effect of phosphate buffer on the enzyme stability was compared to HEPES buffer. Low concentrations of phosphate buffer showed a positive effect on the enzyme initial activity, while higher phosphate buffer concentrations prevented cofactor dissociation. Additionally, the effects of various amine or ketone substrates on the enzyme stability were explored. All tested amines caused a concentration dependent enzyme inactivation, while the corresponding ketones showed no or stabilizing effects. The enzyme inactivation due to the presence of amine can be connected to the formation of PMP, which forms in the presence of amines in the absence of ketone. Since PMP is not covalently bound to the enzyme, it could readily leave the enzyme upon formation. Exploring the different stability effects of cofactor, substrates, additives and buffer system on ATAs seems to be important in order to understand and improve the general performance of ATAs.
  •  
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