| 1. |
- Bloch, K, et al.
(författare)
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Functional activity of insulinoma cells (INS-1E) and pancreatic islets cultured in agarose cryogel sponges
- 2005
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Ingår i: Journal of Biomedical Materials Research. Part A. - : Wiley. - 1552-4965 .- 1549-3296. ; 75A:4, s. 802-809
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Tidskriftsartikel (refereegranskat)abstract
- Here, we describe the preparation, structure, and properties of cryogel sponges, which represent a new type of macroporous biomaterial for tissue engineering. Cryogels were produced through freeze-thawing techniques, either from agarose alone or from agarose with grafted gelatin. The aim of this study was to evaluate agarose cryogel sponges as scaffolds for Culturing both isolated pancreatic islets and insulinoma cells (INS-IE). In order to evaluate the effect of cell entrapment in artificial scaffolds, cell function reflected by insulin secretion and content was studied in cells cultivated for a 2-week period either in Culture plastic plates or in cryogel sponge disks. Our results show that tumor-derived INS-1E cells grown either on plastic or on cryogels do not differ in their proliferation, morphology, insulin release, and intracellular insulin content. However, isolated pancreatic islets cultivated on cryogels sponge show 15-fold higher basal insulin secretion at 3.0 mM glucose than islets cultivated on plastic plates and fail to respond to stimulation with 16.7 mM glucose. In addition, these islets have about 2-fold lower insulin content compared to those grown in plastic plates. It is possible that the cell dysfunction noted in these in vitro experiments is due to the effect of the limited oxygen supply to the islets cultivated in cryogel sponge. Further in vivo Studies are needed to clarify the nature of such an observation since according to previous reports, agarose and gelatin induce new vessel formation supporting enhanced oxygen supply. (c) 2005 Wiley Periodicals, Inc.
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| 2. |
- Dainiak, Maria, et al.
(författare)
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Cell chromatography: Separation of different microbial cells using IMAC supermacroporous monolithic columns
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:2, s. 644-649
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Tidskriftsartikel (refereegranskat)abstract
- Supermacroporous monolithic columns with CU2+-IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross-linked with methylenebis(acrylamide) and has a network of large (10- 100 mu m) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild-type Escherichia coli (w.t. E. coli) and recombinant E. coli cells displaying poly-His peptides (His-tagged E. coli) and of w.t. E. coli and Bacillus halodurans cells. Wild-type E. coli and His-tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8-fold enriched with His-tagged E. coli cells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. coli cells. Haloalkalophilic B. halodurans cells had low affinity to the CU2+-IDA cryogel column and could be efficiently separated from a mixture with w.t. E. coli cells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.
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| 3. |
- Dainiak, Maria, et al.
(författare)
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Improved methods for prepurification and detection of Staphylococcal Enterotoxin B from cell-free culture filtrate
- 2005
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 21:4, s. 1347-1351
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Tidskriftsartikel (refereegranskat)abstract
- An improved ELISA method for the detection of Staphylococcal. Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.
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| 4. |
- Deraz, Sahar, et al.
(författare)
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Purification and characterisation of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079
- 2005
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 117:4, s. 343-354
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriocins are natural antimicrobial agents produced by food fermentative bacteria. Lactobacillus acidophilus DSM 20079 produces a small bacteriocin, with a molecular mass of 6.6 kDa, designated acidocin D20079. This antimicrobial peptide was extremely heat-stable (30 min at 121 degrees C) and was active over a wide pH range. It was found to be sensitive to proteolytic enzymes (trypsin, ficin, pepsin, papain, and proteinase K). Acidocin D20079 has a narrow inhibitory spectrum restricted to the genus Lactobacillus which includes L. sakei NCDO 2714, an organism known to cause anaerobic spoilage of vacuum-packaged meat. Maximum production of acidocin D20079 in MRS broth was detected at pH 6.0, and the peptide was purified by ammonium sulphate precipitation followed by sequential cation exchange and hydrophobic interaction chromatography. Purified acidocin D20079 spontaneously formed spherulite crystals during dialysis. As the N-terminus was found to be blocked for sequencing, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to determine a partial sequence. and the molecular mass of the bacteriocin in the formed crystals (6.6 kDa). Estimates of the molecular weight of the partially purified peptide, using tricine-SDS-PAGE, in which bacteriocin activity was confirmed by overlayer techniques were in accordance with this value. (c) 2005 Published by Elsevier B.V.
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| 5. |
- Galaev, Igor, et al.
(författare)
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High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1065:2, s. 169-175
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Tidskriftsartikel (refereegranskat)abstract
- Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples. (C) 2005 Elsevier B.V. All rights reserved.
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| 6. |
- Hagström, Anna, et al.
(författare)
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Wax esters produced by solvent-free energy-efficient enzymatic synthesis and their applicability as wood coatings
- 2005
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Ingår i: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9270 .- 1463-9262. ; 7:12, s. 837-843
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Tidskriftsartikel (refereegranskat)abstract
- The study aimed at developing a process for making a wood coating wax based on the principles of green chemistry. The research was conducted within the Swedish interdisciplinary research programme Greenchem. Wax esters are attractive since they are non-hazardous, biodegradable and can be produced in an atom-efficient process from building blocks obtained from renewable resources. Four wax esters were prepared in a solvent-free process using an immobilised lipase as catalyst. When the water was removed during the process from what was initially an equimolar mixture of the starting materials carboxylic acid and alcohol by a stream of dry air passed through the reactor, there was a 95-99% conversion to the ester. The enzymatic process consumed 34% less energy and generated less waste than chemical esterification using a strong acid as catalyst. Two of the esters worked well in the industrial wood coating equipment employed and produced surfaces resistant to water and somewhat less to fat stains.
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| 7. |
- Hanora, Amro, et al.
(författare)
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Capture of bacterial endotoxins using a supermacroporous monolithic matrix with immobilized polyethyleneimine, lysozyme or polymyxin B
- 2005
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 118:4, s. 421-433
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial endotoxins (BEs) are integrated part of Escherichia coli, a microorganism widely used for the production of recombinant proteins. BEs should be eliminated in the course of down stream processing of target protein produced by these bacteria. Supermacroporous monolith (continuous bed) columns, so called cryogel columns, with immobilized polyethyleneimine (PEI), polymyxin B (PMB) and lysozyme were employed for BEs capture. Due to the large interconnected pores it was possible to use cryogel columns at flow rates as high as 10 ml/min. The columns packed with Sepharose CL-4B with immobilized PEI, PMB and lysozyme were impossible to use at these high flow rates due to the collapse of the bed. The dynamic capacities of the cryogel columns were nearly independent of the flow rate. In the presence of EDTA, BEs were quantitatively captured from mixtures with a model protein, bovine serum albumin (BSA) at pH 7.2 with practically no protein losses. At pH 3.6 BEs were captured directly from non-clarified E. coli cell lysate resulting in more than 104 times BEs clearance. (C) 2005 Elsevier B.V. All rights reserved.
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| 8. |
- Hanora, Amro, et al.
(författare)
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Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format
- 2005
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1087:1-2, s. 38-44
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Tidskriftsartikel (refereegranskat)abstract
- A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries. (c) 2005 Elsevier B.V. All rights reserved.
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| 9. |
- Hashim, Suhaila, et al.
(författare)
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Alkaline active maltohexaose-forming α-amylase from Bacillus halodurans LBK 34
- 2005
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 36:1, s. 139-146
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Tidskriftsartikel (refereegranskat)abstract
- The gene encoding Amy 34, a maltohexaose-forming α-amylase from Bacillus halodurans LBK 34 isolated from Lake Bogoria, Kenya, was cloned and sequenced. The mature peptide consists of 958 amino acids with a theoretical molecular weight of 107.2 kDa and pI 4.41, respectively. The gene was expressed in Escherichia coli and the recombinant enzyme purified to homogeneity by a combination of metal chelate affinity and size exclusion chromatography. The pure enzyme exhibited optimum activity at 60 °C and pH 10.5–11.5. The enzyme retained over 60% activity after incubation at 55 °C for 4 h and was most stable at pH 9.0. Complete inhibition of enzyme activity was observed in presence of 5 mM Cu2+, Fe2+, Fe3+, Mn2+ and 5 mM EDTA. The enzyme displayed 80% of its original activity in presence of 1% (w/v) SDS and was stable in presence of up to 5 mM DTT. Maltohexaose (G6) was the main initial product of starch hydrolysis while other products formed were G4 > G2 > G5 > G3 and G1. The main end product of the enzyme's action on amylose, amylopectin and maltodextrin is maltotetraose. Amy 34 could not hydrolyse pullulan, α and β-cyclodextrin but could hydrolyse γ-cyclodextrin to produce glucose, maltose and maltotetraose. Maltotetraose was the smallest α-(1–4) linked maltooligosaccharide that could be hydrolysed by the enzyme.
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| 10. |
- Hashim, Suhaila, et al.
(författare)
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Differential scanning calorimetric studies of a Bacillus halodurans alpha-amylase
- 2005
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Ingår i: Biochimica et Biophysica Acta. General Subjects. - : Elsevier BV. - 0304-4165. ; 1723:1-3, s. 184-191
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Tidskriftsartikel (refereegranskat)abstract
- The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T-m) of 70.8 degrees C at PH 9.0, with four transitions, as determined using multiple Gaussian curves. The T-m increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1 - to 5-fold molar excess resulted in an increase in calorimetric enthalpy (Delta H-cal), however, at higher concentrations of CaCl2 (up to 100-fold), Delta H-cal was found to decrease, accompanied by a decrease in entropy change (Delta S), while the T., steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T-m by 10.4 degrees C. T-m was also decreased to 61.1 degrees C and 65.9 degrees C at PH 6.0 and PH 11.0, respectively. (c) 2005 Elsevier B.V. All rights reserved.
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