Thermosensitive, Reversibly Cross-Linking Gels with a Shape "Memory"
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
B Mattiasson
Publications and source records attributed to B Mattiasson.
Interaction of antibodies and antigens conjugated with synthetic polyanions: on the way of creating an artificial chaperone.
Recently we have initiated the use of synthetic polyelectrolytes to mimic the action of chaperones in living cells [Dainiak et al., Biochim. Biophys. Acta 1381 (1998) 279-285]. The next step in this direction is done by the synthesis of conjugates of poly(methacrylic acid) (PMAA) with antigen, denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), and with monoclonal antibodies specific for dGAPDH (but not for the native protein). The pH-dependent properties of the conjugates have been studied using turbidimetry and light scattering. The antibody-PMAA and dGAPDH-PMAA conjugates were shown to interact with free dGAPDH and antibodies respectively as well as with each other. Insoluble aggregates of dGAPDH with antibody-PMAA and of antibodies with dGAPDH-PMAA are formed in acidic media. The same situation occurs in the mixture of antibody-PMAA and dGAPDH-PMAA: precipitation takes place in acidic media, whereas soluble associates are formed in neutral solutions. The size of the soluble associates and the number of conjugates in the associate could be regulated by pH. The competition of free dGAPDH and dGAPDH-PMAA for binding with antibody-PMAA and the dynamic release of refolded GAPDH, with no affinity to antibody-PMAA, into solution could be used for simulating chaperone action.
Anions↗
Polymer displacement/shielding in protein chromatography.
An overview of different applications of polymer interactions with ion-exchange and dye-affinity chromatographic matrices is presented here. The strength of interaction between the ligand and the polymer plays a crucial role in deciding the mode of chromatographic application. Charged, non-ionic and thermosensitive polymers such as poly(ethylene imine), poly(N-vinyl pyrrolidone) and poly(vinyl caprolactam) respectively, show different degrees of interaction with the dye molecules in dye ligand chromatography. Polymers, with their ability of multipoint and hence strong attachment to the chromatographic matrices, were used as efficient displacers in displacement chromatography. The polymer displacement resulted in better recoveries and sharper elution profiles than traditional salt elutions. The globule-coil transition of the thermosensitive reversible soluble-insoluble polymer, poly(vinyl caprolactam), can be exploited in dye-affinity columns for the temperature induced displacement of the bound protein. In another situation, prior to the column chromatography of crude protein extract, polymers formed complexes with the dye matrix and "shielded" the column. The polymer shielding decreased the nonspecific interactions without affecting the specific interactions of the target protein to the dye matrix.
Chromatography↗
Redox hydrogel-based amperometric bienzyme electrodes for fish freshness monitoring
This work presents the design and optimization of amperometric biosensors for the determination of biogenic amines (e.g., histamine, putrescine, cadaverine, tyramine, cystamine, agmatine, spermidine), commonly present in food products, and their application for monitoring of freshness in fish samples. The biosensors were used as the working electrodes of a three-electrode electrochemical cell of wall-jet type, operated at -50 mV vs Ag/AgCl, in a flow injection system. Two different bienzyme electrode designs were considered, one based on the two enzymes [a newly isolated and purified amine oxidase (AO) and horseradish peroxidase (HRP)] simply adsorbed onto graphite electrodes, and one when they were cross-linked to an Os-based redox polymer. The redox hydrogel-based biosensors showed better biosensors characteristics, i.e., sensitivity of 0.194 A M-1 cm-2 for putrescine and 0.073 A M-1 cm-2 for histamine, and detection limits (calculated as three times the signal-to-noise ratio) of 0.17 microM for putrescine and 0.33 microM for histamine. The optimized redox hydrogel-based biosensors were evaluated in terms of stability and selectivity, and were used for the determination of total amine content in fish samples kept for 10 days in different conditions.
Journal Article↗
Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes.
A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.
Animals↗
Degradation of acenaphthene, phenanthrene and pyrene in a packed-bed biofilm reactor.
Biofilm reactors are particularly suitable for the treatment of large amounts of diluted effluent, such as groundwater contaminated with scarcely soluble pollutants. A packed-bed column reactor was tested for the degradation of acenaphthene, phenanthrene and pyrene provided at their aqueous solubility concentrations. Acenapthene and phenanthrene were removed to more than 99% efficiency from this reactor whilst pyrene was removed to 90%. Pollutant disappearance was also recorded in the control reactor and was probably caused by the adsorption of pollutants into the reactor. The measurement of oxygen consumption in both reactors confirmed that microbial degradation of the pollutants was indeed occurring in the inoculated reactor. Physical adsorption is not however unwanted, as it could help with the formation of a biofilm at an early stage of the treatment.
Acenaphthenes↗
Evaluation of parameters for monitoring an anaerobic co-digestion process.
The system investigated in this study is an anaerobic digester at a municipal wastewater treatment plant operating on sludge from the wastewater treatment, co-digested with carbohydrate-rich food-processing waste. The digester is run below maximum capacity to prevent overload. Process monitoring at present is not extensive, even for the measurement of on-line gas production rate and off-line pH. Much could be gained if a better program for monitoring and control was developed, so that the full capacity of the system could be utilised without the risk of overload. The only limit presently set for correct process operation is that the pH should be above 6.8. In the present investigation, the pH was compared with alkalinity, gas production rate, gas composition and the concentration of volatile fatty acids (VFA). Changes in organic load were monitored in the full-scale anaerobic digester and in laboratory-scale models of the plant. Gas-phase parameters showed a slow response to changes in load. The VFA concentrations were superior for indicating overload of the microbial system, but alkalinity and pH also proved to be good monitoring parameters. The possibility of using pH as a process indicator is, however, strongly dependent on the buffering capacity. In this study, a minor change in the amount of carbohydrates in the substrate had drastic effects on the buffering effect of the system.
Anaerobiosis↗
Bacterial metal-resistance proteins and their use in biosensors for the detection of bioavailable heavy metals.
We have expressed and purified metal-resistance and metal regulatory proteins from the bacterial determinants of resistance to heavy metals and utilised these in the development of biosensors for heavy metals. Both the metallothionein from the cyanobacterium Synechococcus PCC 7942 and the MerR regulatory protein from transposon Tn501 allow the detection of non-specific metal binding down to 10(-15) M concentrations of Hg(II), Cu(II), Zn(II) and Cd(II) in pure solution. Differential effects of the metals can be detected at both low and high concentrations, and the shape of the capacitance curves may reflect biologically relevant responses of the proteins to metals. Further work is required to establish the relationship between the detected binding of metal and the biological response of the protein, or to provide biosensors of use in the natural environment.
Bacterial Proteins↗
Quantification of nisin in flow-injection immunoassay systems.
A monoclonal-antibody-based, sequential competitive-flow-injection immunoassay system in expanded-bed mode has been developed for the determination of nisin. The system allows the determination of nisin in the presence of suspended particles without any significant interference, illustrating its potential for on-line monitoring of fermentation processes or the analysis of food matrices. The dose response range of the system when operated in expanded-bed mode was 6-90 microM. The detection limit under packed-bed conditions was 3 microM. The results correlated well with the results from conventional ELISA in the analysis of samples of processed cheese. When milk samples, fermentation samples and buffer were spiked with nisin, the mean recoveries were 86% for milk samples, 96% for fermentation samples and 98% for buffer solution.
Animals↗
Immobilised activated sludge based biosensor for biochemical oxygen demand measurement.
A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4-8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD5 analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below +/- 5.6%, standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. +/- 15% standard deviation.
Biosensing Techniques↗
Superporous agarose monoliths as mini-reactors in flow injection systems. On-line monitoring of metabolites and intracellular enzymes in microbial cultivation processes.
A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15 x 5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 microm in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 A) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min(-1)). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular beta-galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against beta-galactosidase).
Bioreactors↗
Aqueous polymer two-phase systems formed by new thermoseparating polymers.
A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C14) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K = 0.8 to K = 5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.
Animals↗
Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100.
An IgG1 monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.
Antibodies, Monoclonal↗
Affinity chromatography of neoglycoproteins.
Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris-HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.
Boron Compounds↗
Lactic acid production by immobilized Lactobacillus casei in recycle batch reactor: a step towards optimization.
Different nutritional and process parameters influencing lactic acid production by Lactobacillus casei, adsorbed to Poraver beads in a recycle batch reactor system, were studied in an attempt to set up a system having a long operational lifetime and permitting use of high substrate concentrations for maximal conversion to the product. The presence of lactose, even as a minor fraction of the total sugar amount, was necessary for complete utilization by the organism for growth and conversion to lactate. Hydrolysed whey protein constituted a richer source of nitrogen compared to yeast extract. Addition of lactate to the medium at the start of the process resulted in severe inhibition compared with the normal process. For a homofermentative process, pH 6.0 was found to be optimal. The overall productivity of the recycle system was higher under all conditions studied in comparison with the batch process using free cells. Enhancement in productivity in the recycle batch reactor was also accompanied by an increase in density of suspended cells. However, the contribution of the suspended cells to the overall reactor productivity was not noticeable. The bead size of the matrix was found to be important for operational stability of the reactor.
Hydrogen-Ion Concentration↗
Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones.
The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2. 5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50 degrees C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (DeltaSdouble dagger) was the dominating factor. Large negative DeltaSdouble dagger values caused low reaction rates at low aw. When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in DeltaSdouble dagger. In the comparison between hexane and pure water, DeltaGdouble dagger values were higher in hexane due to higher DeltaHdouble dagger (activation enthalpy) values. The enantioselectivity (E value) increased from 2.6 at water activity 0. 53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones.
Alcohol Dehydrogenase↗
Monitoring of low concentrations of glucose in fermentation broth.
A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.
Biosensing Techniques↗
Fast remediation of coal-tar-related compounds in biofilm bioreactors.
The biological degradation of complex mixtures of recalcitrant substances is still a major challenge in environmental biotechnology and the remediation of coal-tar constitutes one such problem area. Biofilm bioreactors offer many advantages and may be successfully used for this purpose. Two stirred-tank reactors and one packed-bed reactor were tested in a continuous mode. Continuous cultivation allows microbial selection to take place whilst adhesive growth provides a high degradation capacity and process stability. The reactors were inoculated with mixed microbial populations to favour complete metabolism and to prevent metabolite accumulation and substrate inhibition effects. Phenol, o-cresol, quinoline, dibenzofuran, acenaphthene and phenanthrene were used as model contaminants and constituted the sole energy and carbon sources. The hydraulic retention time (HRT) was initially set to 2.5 days for a period of several months to allow the establishment of a stable biofilm and was then gradually decreased. All the compounds were found to be degraded by more than 90% at HRT of 3 h or more. Neither substrate inhibition nor metabolite accumulation effects were observed. The stirred-tank configuration was found to be the most efficient for use with high loads. No improvement in the degradation capacity could be achieved by increasing the biofilm surface in these reactors, illustrating that the limiting factor may be the mass transfer limitations rather than the availability of the biofilm surface. Finally, anaerobic treatment was successfully achieved, confirming the potential for remediation of contaminated sites under anaerobic conditions, providing that alternative electron acceptors are present.
Acenaphthenes↗