| 1. |
- 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.
|
|
| 2. |
- Basselet, Pascal, et al.
(författare)
-
Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)
- 2008
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. Results: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2x), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes. Conclusion: In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.
|
|
| 3. |
|
|
| 4. |
- Bollok, Monika, et al.
(författare)
-
Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
- 2005
-
Ingår i: Applied Biochemistry and Biotechnology. - : Springer Science and Business Media LLC. - 0273-2289 .- 1559-0291. ; 126, s. 61-77
-
Tidskriftsartikel (refereegranskat)abstract
- The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.
|
|
| 5. |
- Castan, A., et al.
(författare)
-
Characteristics of a DO-controlled fed-batch culture of Escherichia coli
- 2000
-
Ingår i: Bioprocess engineering (Berlin. Print). - : Springer Science and Business Media LLC. - 0178-515X .- 1432-0797 .- 1615-7591. ; 22:6, s. 509-515
-
Tidskriftsartikel (refereegranskat)abstract
- The DO-controlled glucose limited fed-batch technique was investigated in an E. coli process for production of a recombinant protein. The k(L)ac* value (oxygen transfer rate at zero oxygen concentration) was calculated from on-line gas analysis data during the process. In the investigated processes with induced production of recombinant protein, the k(L)ac* value decreased drastically several hours after induction. The reason for the decrease was found in increasing concentrations of DNA in the medium and increased viscosity due to cell lysis. The consequences of such a dramatic decrease in the volumetric oxygen transfer coefficient on the glucose feed and specific rates are described in computer simulations and experimental data.
|
|
| 6. |
- Castan, A., et al.
(författare)
-
Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli
- 2002
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 77:3, s. 324-328
-
Tidskriftsartikel (refereegranskat)abstract
- Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.
|
|
| 7. |
- Castan, A., et al.
(författare)
-
Oxygen enriched air supply in Escherichia coli processes : production of biomass and recombinant human growth hormone
- 2002
-
Ingår i: Enzyme and microbial technology. - 0141-0229 .- 1879-0909. ; 30:7, s. 847-854
-
Tidskriftsartikel (refereegranskat)abstract
- In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.
|
|
| 8. |
- Castan, A., et al.
(författare)
-
The use of flow cytometry to detect nucleic acids attached to the surface of Escherichia coli in high cell density fed-batch processes
- 2002
-
Ingår i: Biotechnology letters. - 0141-5492 .- 1573-6776. ; 24:3, s. 219-224
-
Tidskriftsartikel (refereegranskat)abstract
- E. coli was grown in an aerobic fed-batch process for the production of a recombinant protein (rhGH). The cells were examined by flow cytometry and PI (propidium iodide) staining. The fluorescence of the PI-stained cells increased with increasing concentrations of DNA in the medium. Furthermore, DNA and RNA attached to the cell could partly be degraded with DNase/RNase and the fluorescence decreased. Formate excretion during the aerobic processes may be due to DNA and possibly also RNA attached to the cell surface, so creating diffusion resistance.
|
|
| 9. |
- Charoenrat, Theppanya, et al.
(författare)
-
Increased total air pressure versus oxygen limitation for enhanced oxygen transfer and product formation in a Pichia pastoris recombinant protein process
- 2006
-
Ingår i: Biochemical engineering journal. - : Elsevier BV. - 1369-703X .- 1873-295X. ; 30:2, s. 205-211
-
Tidskriftsartikel (refereegranskat)abstract
- Two strategies to increase the productivity of secreted Thai Rosewood beta-glucosidase in Pichia pastoris processes were evaluated. Both methods were based on increasing the oxygen transfer rate (OTR) in the process by simple means. Increasing the driving force for the diffusion from the air bubbles to the medium by elevating the air pressure, from 1.2 to 1.9 bar increased the oxygen uptake rate (OUR) by 59% while increasing the driving force by accepting oxygen limitation increased the OUR by 35%. The OTR increased less than in proportion to the increased solubility in the high-pressure process, which indicates that air bubble compression reduces the volumetric oxygen transfer coefficient (K(L)a). Even though the methanol consumption increased almost in proportion to the OTR in both processes the biomass production did not increase as much. This is explained as a higher maintenance demand for methanol in the oxygen limited (0.027 g g(-1) g(-1)) and high-pressure processes (0.035 g g(-1) g(-1)), compared to 0.022 g g(-1) g(-1) in the methanol limited reference process. However, in spite of the low effect of increasing OTR on the biomass production the total beta-glucosidase yield increased almost in proportion to the increased methanol consumption and reached highest value in the high-pressure process, while the beta-glucosidase purity was highest in the oxygen-limited process due to release of less contaminating proteins.
|
|
| 10. |
- Charoenrat, Theppanya, et al.
(författare)
-
Oxygen-limited fed-batch process : an alternative control for Pichia pastoris recombinant protein processes
- 2005
-
Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 27:6, s. 399-406
-
Tidskriftsartikel (refereegranskat)abstract
- An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.
|
|
| 11. |
|
|
| 12. |
- 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.
|
|
| 13. |
- 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.
|
|
| 14. |
- Gabig-Ciminska, Magdalena, et al.
(författare)
-
Detection of bacteriophage infection and prophage induction in bacterial cultures by means of electric DNA chips
- 2004
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 324:1, s. 84-91
-
Tidskriftsartikel (refereegranskat)abstract
- Infections of bacterial cultures by bacteriophages are common and serious problems in many biotechnological laboratories and factories. A method for specific, quantitative, and quick detection of phage contamination, based on the use of electric DNA chip is described here. Different phages of Escherichia coli and Bacillus subtilis were analyzed. Phage DNA was isolated from bacterial culture samples and detected by combination of bead-based sandwich hybridization with enzyme-labeled probes and detection of the enzymatic product using silicon chips. The assay resulted in specific signals from all four tested phages without significant background. Although high sensitivity was achieved in 4h assay time, a useful level of sensitivity (10(7)-10(8) phages) is achievable within 25 min. A multiplex DNA chip technique involving a mixture of probes allows for detection of various types of phages in one sample.. These analyses confirmed the specificity of the assay.
|
|
| 15. |
- Gabig-Ciminska, Magdalena, et al.
(författare)
-
Electric chips for rapid detection and quantification of nucleic acids
- 2004
-
Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 19:6, s. 537-546
-
Tidskriftsartikel (refereegranskat)abstract
- A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the biorecognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.
|
|
| 16. |
- Gabig-Ciminska, Magdalena, et al.
(författare)
-
Gene-based identification of bacterial colonies with an electric chip
- 2005
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 345:2, s. 270-276
-
Tidskriftsartikel (refereegranskat)abstract
- A method for the identification of bacterial colonies based on their content of specific genes is presented. This method does not depend on DNA separation or DNA amplification. Bacillus cereus carrying one of the genes (hblC) coding for the enterotoxin hemolysin was identified with this method. It is based on target DNA hybridization to a capturing probe immobilized on magnetic beads, followed by enzymatic labeling and measurement of the enzyme product with a silicon-based chip. An hblC-positive colony containing 10(7) cells could be assayed in 30 min after ultrasonication and centrifugation. The importance of optimizing the ultrasonication is illustrated by analysis of cell disruption kinetics and DNA fragmentation. An early endpoint PCR analysis was used to characterize the DNA fragmentation as a function of ultrasonication time. The first minutes of sonication increased the signal due to both increased DNA release and increased DNA fragmentation. The latter is assumed to increase the signal due to improved diffusion and faster hybridization of the target DNA. Too long sonication decreased the signal, presumably due to loss of hybridization sites on the targets caused by extensive DNA fragmentation. The results form a basis for rational design of an ultrasound cell disruption system integrated with analysis on chip that will move nucleic acid-based detection through real-time analysis closer to reality.
|
|
| 17. |
- Gabig-Ciminska, Magdalena, et al.
(författare)
-
Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip
- 2004
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 3, s. 2-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic microorganisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption. Results: A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes. The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes. The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h. The method was also successful when applied directly to unpurified spore and cell extract samples. The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal. Conclusions: The sensitivity, convenience and specificity of the system have shown its potential. In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest. This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification.
|
|
| 18. |
- Han, L., et al.
(författare)
-
Changes in intracellular metabolite pools, and acetate formation in Escherichia coli are associated with a cell-density-dependent metabolic switch
- 2002
-
Ingår i: Biotechnology letters. - 0141-5492 .- 1573-6776. ; 24:6, s. 483-488
-
Tidskriftsartikel (refereegranskat)abstract
- A cell density-dependent metabolic switch in amino acid metabolism occurs in E. coli W3110 batch cultures at 1.15 g dry wt l(-1) (Han L, Doverskog M, Enfors S-O, Haggstrom L, 2002, J. Biotechnol. 92: 237-249). A two- to three-fold decrease of the concentration of most glycolytic and citric acid cycle metabolites, and an increase in acetyl-CoA concentration after the switch, indicates that the central metabolism also is affected. The specific acetate production rate decreases throughout the culture, except for a temporary increase at the switch point. The intracellular acetate concentration remains relatively constant during the culture.
|
|
| 19. |
- 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.
|
|
| 20. |
- Han, L., et al.
(författare)
-
Escherichia coli high-cell-density culture : carbon mass balances and release of outer membrane components
- 2003
-
Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 25:4, s. 205-212
-
Tidskriftsartikel (refereegranskat)abstract
- Carbon mass balances were calculated in fed-batch cultures of E. coli W3110, using mineral medium with glucose as the limiting substrate. The carbon recovery, based on biomass, CO2 and acetate was similar to90% at the end of the culture (25 h, 27 g L-1 dw). The missing carbon remained as soluble organic compounds in the medium. Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at similar to1 g L-1) contributed to 63% of the extracellular carbon. The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture. Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred. Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the carbon mass. The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.
|
|
| 21. |
|
|
| 22. |
- Ignatova, Z., et al.
(författare)
-
The relative importance of intracellular proteolysis and transport on the yield of the periplasmic enzyme penicillin amidase in Escherichia coli
- 2000
-
Ingår i: Enzyme and microbial technology. - 0141-0229 .- 1879-0909. ; 26:04-feb, s. 165-170
-
Tidskriftsartikel (refereegranskat)abstract
- Intracellular proteolysis is an important mechanism for regulating the level of the periplasmic enzyme penicillin amidase in Escherichia coli. Evidence is presented that the active enzyme is localized in the periplasmic space and maturation of pro-enzyme occurs during transport through the cytoplasmic membrane or rapidly after its entrance in the periplasm. The rate constants of the transport through cytoplasmic membrane and of the intracellular proteolysis were estimated to be 0.01 h and 0.5 h, respectively. This indicates that more than 90% of the synthesized pre-pro-enzyme is lost by intracellular proteolysis occurring in the cytoplasm.
|
|
| 23. |
- 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.
|
|
| 24. |
- Jahic, M., et al.
(författare)
-
Interfacing Pichia pastoris cultivation with expanded bed adsorption
- 2006
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 93:6, s. 1040-1049
-
Tidskriftsartikel (refereegranskat)abstract
- For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L (.) h.
|
|
| 25. |
- Jahic, Mehmedalija, et al.
(författare)
-
Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein
- 2002
-
Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 24:6, s. 385-393
-
Tidskriftsartikel (refereegranskat)abstract
- A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O-2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.
|
|
| 26. |
- Jahic, Mehmedalija, et al.
(författare)
-
Process technology for production and recovery of heterologous proteins with Pichia pastoris
- 2006
-
Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 22:6, s. 1465-1473
-
Forskningsöversikt (refereegranskat)abstract
- Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.
|
|
| 27. |
- Jahic, Mehmedalija, et al.
(författare)
-
Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures.
- 2003
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 2:1, s. 6-
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut+ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. RESULTS: A protein concentration of about 1 g L-1 was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L-1 full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40-70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process. CONCLUSION: Three mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity.
|
|
| 28. |
|
|
| 29. |
- Le, T. N., et al.
(författare)
-
Expression and simple purification strategy for the generation of antimicrobial active enterocin P from Enterococcus faecium expressed in Escherichia coli ER2566
- 2014
-
Ingår i: Iranian Journal of Biotechnology. - : Armenian Green Publishing Co.. - 1728-3043 .- 2322-2921. ; 12:4, s. 16-25
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Enterocin-P of Enterococcus faecium P13 (EntP) is of great interest as a food preservative and medicine due to its non-toxicity and broad antimicrobial spectrum at various pH, as well as its excellent thermal stability. However, recombinant production of EntP is still in laboratory because of low productivity and complex purification process. Objectives: In this study, we aimed to develop efficient methods for high-level expression and convenient purification of the recombinant EntP.Materials and Methods: An artificially synthesized gene (entP) of 132 bp encoding mature enterocin P of E. faecium P13 was cloned in plasmid pTWIN1 under the control of an inducible T7lac promoter for expression of fusion protein EntP- Mxe GyrA mini-intein-chitin binding domain (CBD) (abbreviated by EntP-Int-CBD) in E. coli. Recombinant EntP was released from the fusion protein by DTT digestion and cleaned by distilled water and checked for anti-bacterial activity. Results: The fusion protein was highly expressed in insoluble form in E. coli at 37ºC with 0.05 mM IPTG induction. The insoluble fusion protein EntP-Int-CBD was easily prepared by cell sonication and centrifugation to remove soluble con- taminants. The repeat washing steps with Triton X-100 were applied to reduce contaminants. After DTT-induced self- digestion in urea 4 M, the EntP released from the fusion protein was insoluble in water and easier to be separated from sol- uble Int-CBD by centrifugation. The recombinant peptide was soluble in 20% 2-propanol in 0.1% trifluoroacetic acid (TFA) and exhibited strong anti- Listeria monocytogenes and Staphylococcus aureus activities.Conclusions: This study is the first report providing a simple, quick and straight forward procedure for heterologous production of functional and pure Enterocin P without using any chromatography columns in the purification process.
|
|
| 30. |
- Lin, H. Y., et al.
(författare)
-
Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli
- 2004
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 87:5, s. 602-613
-
Tidskriftsartikel (refereegranskat)abstract
- Guanosine-3'5'-tetraphosphate (ppGpp) and as, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.
|
|
| 31. |
- Lin, H. Y., et al.
(författare)
-
Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli
- 2001
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 73:5, s. 347-357
-
Tidskriftsartikel (refereegranskat)abstract
- A simple pulse-based method for the determination of the maximum uptake capacities for glucose a nd oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where mu was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when mu declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.
|
|
| 32. |
- Liu, Yanling, 1974-, et al.
(författare)
-
Confirmative electric DNA array-based test for food poisoning Bacillus cereus
- 2007
-
Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 70:1, s. 55-64
-
Tidskriftsartikel (refereegranskat)abstract
- Detection of the full set of toxin encoding genes involved in gastrointestinal diseases caused by B. cereus was performed. Eight genes determining the B. cereus pathogenicity, which results in diarrhea or emesis, were simultaneously evaluated on a 16-position electrical chip microarray. The DNA analyte preparation procedure comprising first 5 min of ultrasonic treatment, DNA extraction, and afterwards an additional 10 min sonication, was established as the most effective way of sample processing. No DNA amplification step prior to the analysis was included. The programmed assay was carried out within 30 min, once the DNA analyte from 10(8) bacterial cells, corresponding to one agar colony, was subjected to the assay. In general, this work represents a mature analytical way for DNA review. It can be used under conditions that require almost immediate results.
|
|
| 33. |
- Liu, Yanling, 1974-, et al.
(författare)
-
Critical factors for the performance of chip array-based electrical detection of DNA for analysis of pathogenic bacteria
- 2008
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 382:2, s. 77-86
-
Tidskriftsartikel (refereegranskat)abstract
- Different factors influencing chip array-based electrical detection of DNA for analysis of pathogenic bacteria were examined. Both rehydration of capture probe layer of functionalized chip arrays and efficient hybridization of targets irrespective of their length resulted in signal enhancement when high-ionic phosphate-buffered saline (i.e., 600 mM sodium chloride and 40 mM disodium hydrogen phosphate) was used. Similarly, placement of two adjacent capture and detection probe-binding sites at a terminal part of the target strand resulted in significant signal increase. Moreover, 10-min ultrasonic fragmentation of targets amplified the signals Lip to twofold for longer DNA strands (i.e., >300 bp). No obvious effects on signals were visible for shorter than 400-bp PCR amplicons subjected to ultrasonication. For DNA strands of all sizes, more than 10 min ultrasonication diminished the specific electrical responses. Our results also demonstrate that target analytes are detected with discrimination against mismatches even for single nucleotide sequence alteration. The mismatch detection appeared in order of ease Of recognition as follows: triple random > quintuple middle > triple middle > single middle mismatch. Among the three variants of one-base mismatches, a sequence variation was most remarkable for adenine. On the other hand, no benefits in assay sensitivity were recognized by the use of longer capture probe linkers as the 6-C linker.
|
|
| 34. |
- Liu, Yanling
(författare)
-
Electric DNA arrays for determination of pathogenic Bacillus cereus
- 2007
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Silicon-based electric chip arrays were developed for characterization of Bacillus cereus with respect to the capacity to produce toxins involved in food poisoning and foodborne infections. Bacteria of the B. cereus group contain different sets of four toxins encoded by eight genes. The purpose of this work was to develop a fast method for determination of the presence of these genes in colonies from primary enrichment cultures. The specific DNA detection was based on immobilization of DNA capture probes, which hybridize to specific sites on the target genes. Biotin-labeled detection probes were designed to hybridize with the target DNA adjacent to the capture probes. An extravidin - alkaline phosphatase complex was subsequently bound to the hybridized detection probes. Finally, p-aminophenyl phosphate was added as substrate for the enzyme, and the product p-aminophenol was brought in contact with the interdigitated gold electrode on the silicon chips surface. The p-aminophenol was oxidized at the anode to quinoneimine, which was then reduced back to paminophenol at the cathode. This redox recycling generates a current that was used as the DNA-chip response to the target DNA. Two versions of the assay were used. In the first version the capture probes were immobilized on magnetic beads and all chemical reactions until and including the enzymatic reaction took place in an eppendorf tube while the redox recycling was used to measure the amount of paminophenol produced after transfer from the tube to the silicon chip surface. In the second version a silicon chip array was used with 16 parallel electrode positions, each activated by immobilization of one type of capture probes on the gold electrodes. With this system all chemical reactions took place at the chip surface. The kinetics of cell disruption and DNA fragmentation from B. cereus by ultrasonication was determined. Maximum cell disruption was achieved within 5 min and the chip response increased in proportion to the ultrasonic time. Further ultrasonication up to 10 min resulted in further increasing current although no further cell disruption was observed. If the sonication time was extended above 10 min the signal declined. Based on analysis of the DNA size distribution by early end-point PCR and gel electrophoresis, it is suggested that the first 5 min ultrasonication increased the signal by increasing the release of target DNA molecules. Thereafter the signal was increased by fragmentation of target DNA which increases the diffusion rate and also the accessibility of the hybridization site. Finally, the DNA fragment sizes approached that of the hybridization site (51-bp) which may reduce the signal because of cleavage of the target DNA in the hybridization region. These studies were performed with the bead-based hybridization assay. The assay was highly specific to the target gene (hblC) of both B. cereus and B. thuringiensis with no response from negative control cells of B. subtilis. The 16 positions of the silicon chip array were activated by immobilization of all known toxin-coding genes of B. cereus and also included both a positive control and a negative control electrode positions. When these chips were exposed to ultrasonicated B. cereus, the gold electrodes were fouled by some component in DNA cell lysates. To circumvent this, the released large DNA was first extracted and then ultrasonicated again, since the extract mainly contains large molecular weight DNA. This DNA extract was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the DNA chip arrays. The results agreed with PCR control analysis which means that these electric DNA chip arrays can be used to characterize bacterial colonies with respect to the genes coding of all known toxins of B. cereus: haemolysin (hblA, hblC, hblD), non-haemolytic enterotoxin (nheA, nheB, nheC), cytotoxin K-2 (cytK-2), and cereulide (ces). The chip assay required about 30 min after application of DNA samples. Due to the generic properties of the chips, this technique should also be applicable for characterization of the pathogenicity potential of many other organisms. Keywords: Bacillus cereus, haemolysin, non-haemolytic enterotoxin, cytotoxin K-2, cereulide, toxin-coding genes, bacterial colony, electric DNA chip, ultrasonication, DNA fragmentation.
|
|
| 35. |
- Liu, Yanling, 1974-
(författare)
-
Electric DNA chips for determination of pathogenic microorganisms
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Silicon-based electric DNA chip arrays were utilized to fast identify pathogenic microorganisms with respect to the capacity to produce toxins involved in foodborne poisoning and infections. Bacteria of the B. cereus and the enterohemorrhagic E. coli (EHEC) groups contain different set-ups of various virulence factors that are encoded by the corresponding genes. The purpose of this work was to develop a fast and simple method for determination of the presence of these virulence genes in a colony from primary enrichment cultures. A target gene is detected through hybridization to a surface-immobilized specific capture probe and biotin-labeled detection probe. Following binding of an enzyme conjugate to this sandwich hybrid complex, a current signal is generated by electronic redox recycling of the enzymatic product paminophenol (pAP). Two versions of the assay were developed. In the first version the capture probes were immobilized on magnetic beads, which carried out all reactions until the pAP generation, while the final electric signal was created by transferring pAP to a single-electrode chip surface. In the second version a silicon chip array with 16 parallel sensing electrode positions each of them functionalized by capture probes, carried out all assay steps on the chip surface. This instrument can realize automatic and multiplexed gene detection. The kinetics of bacterial cell disruption and impact of DNA fragmentation by ultrasound were determined. The experimental data suggested that the increased signal after first minutes of ultrasonication were due to the accumulation of released DNA amount, while the further signal increase resulted from the improved hybridization with the shortened target DNA strands. Studies on probe localization on the 16-electrode chip assays indicated that the probe-targeting site, which was located at the 5’-end of strands, gave rise to the highest signal level due to the efficient targetprobes hybridization and the following enzyme binding. When these functionalized chip arrays were exposed to the cell homogenates, the sensing electrodes were fouled by cellular proteins and therefore led to dramatically decreased redox-recycling current. To circumvent this, samples were treated by DNA extraction after the 1st sonication and then DNA fragmentation by a 2nd time sonication. The DNA extract removed most of the interfering components from bacterial cell. This sample treatment was applied to characterize one “diarrheal” and one “emetic” strain of B. cereus with the chip arrays functionalized by eight DNA probes. The signal patterns of eight virulence genes from chip assays agreed well with PCR control analyses for both strains. By simply adding the SDS detergent to cell homogenates, chip surface blocking effect can be significantly reduced even without DNA extraction treatment. After optimization of some critical factors, the 16-electrode DNA chips with the improved sensing performance can directly detect multiple virulence genes from a single E. coli colony in 25 min after the introduction of supernatant of ultrasonicated cell lysate.
|
|
| 36. |
|
|
| 37. |
- Rozkov, Aleksei D., et al.
(författare)
-
Analysis and control of proteolysis of recombinant proteins in Escherichia coli.
- 2004
-
Ingår i: Advances in biochemical engineering/biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. ; 89, s. 163-195
-
Tidskriftsartikel (refereegranskat)abstract
- Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli. Important properties of E. coli proteases, which are relevant for the production of recombinant proteins, are reviewed. Furthermore, various strategies to control the proteolysis of the recombinant proteins are presented. These strategies for control of proteolysis can be applied on various stages of the process: design of more stable protein, a modification of the host cell in respect to proteolytic activity, optimisation of cultivation and downstream processing. However, before implementing these measures the proteolysis rate should be measured in order to calculate a potential benefit of reduced proteolysis rate.
|
|
| 38. |
- Rozkov, A., et al.
(författare)
-
Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins
- 2000
-
Ingår i: Enzyme and microbial technology. - 0141-0229 .- 1879-0909. ; 27:10, s. 743-748
-
Tidskriftsartikel (refereegranskat)abstract
- A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg.g(-1)) compared to the fusion protein SpA-beta galactosidase (138 mg.g(-1)) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the Ist order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg.g(-1) to 120 mg.g(-1). The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg.g(-1) in II h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.
|
|
| 39. |
|
|
| 40. |
|
|
| 41. |
|
|
| 42. |
- Sundström, Heléne, 1962-
(författare)
-
Analytical tools for monitoring and control of fermentation processes
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The overall objective of this work has been to adopt new developments and techniques in the area of measurement, modelling and control of fermentation processes. Flow cytometry and software sensors are techniques which were considered ready for application and the focus was set on developing tools for research aiming at understanding the relationship between measured variables and process quality parameters. In this study fed-batch cultivations have been performed with two different strains of Escherichia coli (E.coli) K12 W3110 with and without a gene for the recombinant protein promegapoietin. Inclusion body formation was followed during the process with flow cytometric detection by labelling the inclusion bodies with first an antibody against the protein promegapoietin and then a second fluorescent anti-antibody. The approach to label inclusion bodies directly in disintegrated and diluted cell slurry could be adopted as a method to follow protein production during the process, although the labelling procedure with incubation times and washings was somewhat time-consuming (1.5 h). The labelling of inclusion bodies inside the cells to follow protein production was feasible to perform, although an unexplained decrease in the relative fluorescence intensity occurred late in process. However, it is difficult to translate this qualitative measurement into a quantitative one, since a quantitative protein analysis should give data proportional to the volume, while the labelling of the spheric inclusion bodies gives a signal corresponding to the area of the body, and calibration is not possible. The methods were shown to be useful for monitoring inclusion body formation, but it seems difficult to get quantitative information from the analysis. Population heterogeneity analysis was performed, by using flow cytometry, on a cell population, which lost 80-90% viability according to viable count analysis. It was possible to show that the apparent cell death was due to cells incapable of dividing on agar plates after induction. These cells continued to produce the induced recombinant protein. It was shown that almost all cells in the population (≈97%) contained PMP, and furthermore total protein analysis of the medium indicated that only about 1% of the population had lysed. This confirms that the "non-viable" cells according to viable count by cfu analysis produced product. The software sensors XNH3 and µNH3, which utilises base titration data to estimate biomass and specific growth rate was shown to correlate well with the off-line analyses during cultivation of E. coli W3110 using minimal medium. In rich medium the µNH3 sensor was shown to give a signal that may be used as a fingerprint of the process, at least from the time of induction. The software sensor KLaC* was shown to respond to foaming in culture that probably was caused by increased air bubble dispersion. The RO/S coefficient, which describes the oxygen to substrate consumption, was shown to give a distinct response to stress caused by lowered pH and addition of the inducing agent IPTG. The software sensor for biomass was applied to a highly automated 6-unit multi-bioreactor system intended for fast process development. In this way also specific rates of substrate and oxygen consumption became available without manual sampling.
|
|
| 43. |
- Sundström, Heléne, et al.
(författare)
-
Segregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes
- 2004
-
Ingår i: Biotechnology Letters. - 0141-5492 .- 1573-6776. ; 26:19, s. 1533-1539
-
Tidskriftsartikel (refereegranskat)abstract
- In Escherichia coli fermentation processes, a drastic drop in viable cell count as measured by the number of colony forming units per ml (c.f.u. ml(-1)) is often observed. This phenomenon was investigated in a process for the production of the recombinant fusion protein, promegapoietin (PMP). After induction, the number of c.f.u. ml(-1) dropped to approximately 10% of its maximum though the biomass concentration continued to increase. Flow cytometric analysis of viability and intracellular concentration of PMP showed that almost all cells were alive and contributed to the production. Thus, the drop in the number of c.f.u. ml(-1) probably reflects a loss of cell division capability rather than cell death.
|
|
| 44. |
- Sundström, Heléne, et al.
(författare)
-
Software sensors for fermentation processes
- 2008
-
Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 31:2, s. 145-152
-
Tidskriftsartikel (refereegranskat)abstract
- Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R-O/S sensor which is the ratio between oxygen and energy substrate consumption. The R-O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R-O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.
|
|
| 45. |
- Surribas, Anna, et al.
(författare)
-
Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies
- 2007
-
Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 130:3, s. 291-299
-
Tidskriftsartikel (refereegranskat)abstract
- Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.
|
|
| 46. |
- Svensson, Marie, et al.
(författare)
-
Control of endotoxin release in Escherichia coli fed-batch cultures
- 2005
-
Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 27:2, s. 91-97
-
Tidskriftsartikel (refereegranskat)abstract
- High amounts of outer membrane (OM) components were released in glucose-limited fed-batch (GLFB) cultures at 37 degrees C at specific growth rates approaching 0.05 h(-1). Endotoxin analyses from a 20 degrees C GLFB culture gave similar results. An alternative fermentation technique, the temperature-limited fed-batch (TLFB) technique, reduced the endotoxin concentration in a culture with a biomass concentration of 30 g l(-1) from the 850 mg l(-1) in traditional GLFB cultures to about 20 mg l(-1). The TLFB technique uses the temperature to regulate the dissolved oxygen tension, while all substrate components are unregulated. It appears to be severe glucose limitation that triggers the extensive release of endotoxins rather than a low growth rate. Furthermore, it is not the low temperature that stabilizes the OM when using the TLFB technique. Simulations and experimental data show that this technique results in the same biomass productivity as the GLFB technique.
|
|
| 47. |
|
|
| 48. |
|
|
| 49. |
- Svensson, Marie, et al.
(författare)
-
Osmotic stability of the cell membrane of Escherichia coli from a temperature-limited fed-batch process
- 2005
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 67:3, s. 345-350
-
Tidskriftsartikel (refereegranskat)abstract
- The temperature-limited fed-batch (TLFB) process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. In Escherichia coli cultures, it has been proven to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, that occurs in the glucose-limited fed-batch (GLFB) processes at specific growth rates below 0.1 h(-1). The TLFB and the GLFB process were compared to each other when applied to produce the periplasmic, constitutively expressed, enzyme beta-lactamase. The extraction of the enzyme was performed by osmotic shock. A higher production of beta-lactamase was achieved with the TLFB technique while no difference in the endotoxin release was found during the extraction procedure. Furthermore, it was found that growth at declining temperature, generated by the TLFB technique, gradually stabilizes the cytoplasmic membrane, resulting in a significantly increased product quality in the extract from the TLFB cultures in the osmotic shock treatment.
|
|
| 50. |
- Svensson, Marie, 1972-
(författare)
-
The temperature-limited fed-batch technique for control of Escherichia coli cultures
- 2006
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The objective of this study was to investigate the physiology and productivity in Escherichia colicultures with emphasis on the temperature-limited fed-batch (TLFB) culture. The TLFB techniquecontrols the oxygen consumption rate of the growing culture by a gradually declining temperaturefrom 37-35 °C down to 20-18 °C. The temperature regulated the DOT around a set-point (30 % airsat.), and all nutrients were in excess. Glucose was fed into the culture continuously, however, highacetate formation was avoided by keeping the glucose at a low, yet excessive, concentration. Thebiomass productivity was approximately the same in TLFB as in glucose-limited fed-batch (GLFB)cultures, since the specific growth rate and the oxygen consumption rate are limited by the oxygentransfer capacity of the reactor in both techniques.High concentrations of endotoxins were found in the medium of E. coli fed-batch cultures at lowspecific growth rates (below 0.1 h-1) and severe glucose limitation. In this thesis the TLFB techniquewas found to suppress the endotoxin release even at low specific growth rates. The repressed release of endotoxins in TLFB cultures was due to the high availability of glucose and not to the low growthrate or the lower temperature. The conclusion was drawn from comparing with the GLFB technique performed at 20 °C, which resulted in high endotoxin release.Extensive release of endotoxin, accompanied with high concentrations of soluble proteins was foundin a TLFB culture exposed to a higher energy dissipation rate, 16 kW m-3, instead of 2 kW m-3, due toa higher stirrer speed (1000 instead of 500 rpm). The hypothesis that this is a result of mechanicalstress is discussed in context with the common view that cells like E. coli, which are smaller than the Kolmogoroff’s microscale of turbulence, should not be influenced by the turbulence.TLFB cultured cells exhibited more stable cytoplasmic membranes when treated with osmotic shockas compared to the GLFB cultured cells. The concentrations of DNA and soluble proteins in the periplasmic extracts from the TLFB cultured cells were lower than from GLFB cultured cells. Inaddition, the specific productivity of periplasmic β-lactamase was higher in the TLFB cultures,suggesting that this technique could be an alternative for protein production. The solubility of apartially aggregated recombinant protein increased in the TLFB compared to the GLFB cultures.However some time after induction, in spite of the gradually declining temperature, the solublefraction decreased.For obtaining better understanding of the performance of the process and for identifying criticalparameters, a mathematical model was developed based on the growth, energy and overflowmetabolism at non-limiting nutrient conditions. The temperature-dependent maximum specific glucoseand oxygen uptake rates were determined in pH-auxostat cultures for temperatures ranging from 18 to37 °C. A dynamic simulation model of the TLFB technique was developed and the results were compared to experimental data. The simulation program was also used for sensitivity analysis of some physiological and process parameters to study the impact on biomass concentration and temperatureprofiles. An effect on the biomass concentration profile but not on the temperature profile wasobserved when changing the oxygen transfer coefficient. If the maximum specific glucose uptake ratewas altered, or if the glucose concentration was permitted to assume other values, the temperatureprofile but not the biomass concentration profile was influenced. Cell death affected both the biomassconcentration profile and the temperature profile.
|
|
