| 1. |
- Ahlqvist, Josefin, et al.
(författare)
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Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies
- 2006
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Ingår i: JOURNAL OF BIOTECHNOLOGY. - ELSEVIER SCIENCE BV. - 0168-1656. ; 122:2, s. 216-225
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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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| 2. |
- Ahlqvist, Josefin, et al.
(författare)
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Monitoring the production of inclusion bodies during fermentation and enzyme-linked immunosorbent assay analysis of intact inclusion bodies using cryogel minicolumn plates
- 2006
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Ingår i: ANALYTICAL BIOCHEMISTRY. - ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0003-2697. ; 354:2, s. 229-237
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Tidskriftsartikel (refereegranskat)abstract
- A novel minicolumn chromatgraphic method to monitor the production of inclusion bodies during fermentation and anenzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced its inclusion bodies wits labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous Monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed LIS to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed oil the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary I-G and of enzymatic reaction within the adsorbent. (c) 2006 Elsevier Inc. All rights reserved.
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| 3. |
- Alvarez Fernandez, Marcia, et al.
(författare)
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Enhancement of sulphide production in anaerobic packed bed bench-scale biofilm reactors by sulphate reducing bacteria
- 2006
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Ingår i: BIOTECHNOLOGY LETTERS. - SPRINGER. - 0141-5492. ; 28:3, s. 175-181
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Tidskriftsartikel (refereegranskat)abstract
- Two biofilm reactors, using pumice stone and Poraver as biofilm supports, were run, based on the optimization of sulphide production using a factorial design. The maximum H2S concentrations reached were 10 and 15 mm, respectively, both being appropriate for metal precipitation in effluents. The set-up of the pumice stone biofilm reactor is suitable for application in the mining area in the Bolivian Andean region, where this material is widely available. The use of specific primers for sulphate-reducing bacteria groups permits the identification of the sulphide-producing bacteria present in biofilms.
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| 4. |
- Andac, M, et al.
(författare)
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Poly(hydroxyethyl methacrylate)-based macroporous hydrogels with disulfide cross-linker
- 2008
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Ingår i: Macromolecular Chemistry and Physics. - Wiley-V C H Verlag GMBH. - 1022-1352. ; 209:6, s. 577-584
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Tidskriftsartikel (refereegranskat)abstract
- Biodegradable supermacroporous PHEMA cryogels were produced by combining two cross-linkers, poly(ethylene glycol) diacrylate and a newly developed disulfide water soluble crosslinker, N,N'-bis(methacryloyl)-L-cystine. The biodegradable PHEMA cryogels were prepared with gel fraction yields up to 70% and were characterized by highly interconnected pores of micrometer size and good mechanical stability. When subjected to reductive agents like DTT, the biodegradable PHEMA cryogels disintegrated into small pieces. The rate of disintegration was controlled by the crosslinking density in the cryogels and the DTT concentration.
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| 5. |
- Andersson, B Erik, et al.
(författare)
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Incomplete degradation of polycyclic aromatic hydrocarbons in soil inoculated with wood rotting fungi and their effect on the indigenous soil bacteria.
- 2003
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Ingår i: Environmental Toxicology and Chemistry. - Society of Environmental Toxicology and Chemistry. - 1551-5028. ; 22:6, s. 1238-1243
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Tidskriftsartikel (refereegranskat)abstract
- Soil artificially contaminated with fluorene, phenanthrene, pyrene, and benzaanthracene was inoculated with the wood-rotting fungi Pleurotus ostreatus and Antrodia vaillantii. During 12 weeks of incubation, polycyclic aromatic hydrocarbon (PAH) degradation and the formation of persistent degradation products were monitored by chemical analysis. In addition, the effect on the indigenous soil bacteria was studied by plate count techniques and by measuring the concentration of bacteria-specific phospholipid fatty acids (PLFAs). In both soils inoculated with fungi, the PAH degradation was enhanced compared to the control soil without fungi. The white-rot fungus P. ostreatus accelerated the degradation rate radically the first weeks, while the effect of the brown-rot fungus was more pronounced at later stages during the 12-week study. In a soil with no amendments, the final degradation result was similar to that in the soil with added fungi, although the degradation pattern for the individual PAHs was different. Furthermore, the degradation by P. ostreatus was accompanied by an accumulation of PAH metabolites, that is, 9-fluorenone, benzaanthracene-7,12-dione, and two compounds identified as 4-hydroxy-9-fluorenone and 4-oxapyrene-5-one, that was not seen in the other soils. The inoculation with the white-rot fungus also had a large negative effect on the indigenous soil bacteria. This could be an important drawback of using the white-rot fungus P. ostreatus in soil bioremediation since a sequential fungal–bacterial degradation probably is needed for a complete degradation of PAHs in soil. In the soil inoculated with A. vaillantii, on the other hand, no metabolites accumulated, and no negative effects were observed on the indigenous microorganisms.
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| 6. |
- Andersson, Jonatan, et al.
(författare)
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Isolation of potato proteins using simulated moving bed technology.
- 2008
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Ingår i: Biotechnology and bioengineering. - Wiley Periodicals, Inc. - 1097-0290. ; 101, s. 1256-1263
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Tidskriftsartikel (refereegranskat)abstract
- The simulated moving bed (SMB) concept of chromatography was applied to treat potato juice from production of starch. The aim was to harvest proteins. SMB offers possibilities to operate with different process strategies and in this study it was shown possible to harvest up to 80% of the protein in a process utilizing very little extra water besides that already present in the juice. After depleting protein from the juice in the adsorption step, the flow through was used to recondition the column after elution. The present study illustrates a new concept of applying chromatography as a capturing step of bulk products. Biotechnol. Bioeng. (c) 2008 Wiley Periodicals, Inc.
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| 7. |
- Andersson, Jonatan, et al.
(författare)
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Simulated moving bed technology with a simplified approach for protein purification - Separation of lactoperoxidase and lactoferrin from whey protein concentrate
- 2006
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Ingår i: Journal of Chromatography A. - Elsevier Science B.V.. - 0021-9673. ; 1107:1-2, s. 88-95
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Tidskriftsartikel (refereegranskat)abstract
- Simulated moving bed (SMB) technology is a continuous chromatographic technique proven to have many advantages compared to conventional batch chromatography, such as: raised productivity and product concentration, reduced buffer consumption as well as more efficient use of raw material. In this study a 20 column SMB process for the separation of lactoperoxidase and lactoferrin from whey protein concentrate (WPC) was developed. A simplified approach with data from a single column experiment was used when designing the process. The SMB process data were compared to a theoretical scale-up of the breakthrough experiment reflecting the same 20 column set-up run in non-moving bed mode. The outcome of the comparison is a 48% raise in productivity, a 4.3 times decrease in buffer consumption, 6.5 times raise in target protein concentration with a raw material utilization which is slightly better for the SMB process.
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| 8. |
- Aragao, Rosa, et al.
(författare)
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Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads.
- 2013
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Ingår i: Biotechnology Letters. - Springer. - 1573-6776. ; 35:3, s. 397-405
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Tidskriftsartikel (refereegranskat)abstract
- Alginate microbeads, produced by emulsion/internal gelation, were studied for the entrapment and microcultivation of microbial cells with biotechnological potential. An anaerobic consortium which was selected for its capacity to degrade complex carbohydrates, and a pure culture of cellulose degrading bacteria were used for entrapment studies. Optimization of conditions for the formation of spherical alginate microbeads in sizes between 20 and 80 μm were examined. The best conditions were achieved by combining rapeseed methyl ester as oil phase and stirring at 100 rpm using a rotation impeller. Calcium alginate microbeads produced under these conditions were shown to present morphological stability, with large pores in the internal matrix that favours microcolony development. Finally, single cells were observed inside the beads after the entrapment procedure and microcolony formation was confirmed after cultivation in cellobiose.
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| 9. |
- Arvidsson, Pär, et al.
(författare)
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Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns
- 2002
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Ingår i: Journal of Chromatography A. - Elsevier Science. - 0021-9673. ; 977:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Continuous supermacroporous chromatographic columns with anion-exchange ligands 2-(dimethylanlino)ethyl group and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 mum in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use. (C) 2002 Published by Elsevier Science B.V.
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| 10. |
- Arvidsson, Pär, et al.
(författare)
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Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent
- 2003
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Ingår i: Journal of Chromatography A. - Elsevier. - 0021-9673. ; 986:2, s. 275-290
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Tidskriftsartikel (refereegranskat)abstract
- A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N,N′-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10–100 m size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His)6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 °C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His)6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts
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