| 1. |
- Olsson, L, et al.
(författare)
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Kinetics of ethanol production by recombinant Escherichia coli KO11
- 1995
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 45:4, s. 356-365
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Tidskriftsartikel (refereegranskat)abstract
- The fermentation kinetics for separate as well as simultaneous glucose and xylose fermentation with recombinant ethanologenic Escherichia coli KO11 are presented. Glucose and xylose were consumed simultaneously and exhibited mutual inhibition. The glucose exhibited 15 times stronger inhibition in xylose fermentation than vice versa. The fermentation of condensate from steam-pretreated willow (Salix) was investigated. The kinetics were studied in detoxified as well as in nondetoxified condensate. The fermentation of the condensate followed two phases: First the glucose and some of the pentoses (xylose in addition to small amounts of arabinose) were fermented simultaneously, and then the remaining part of the pentoses were fermented. The rate of the first phase was independent of the detoxification method used, whereas the rate of the second phase was found to be strongly dependent. When the condensate was detoxified with overliming in combination with sulfite, which was the best detoxification method investigated, the sugars in the condensate, 9 g/L, were fermented in 11 h. The same fermentation took 150 h in nondetoxified condensate, The experimental data were used to develop an empirical model, describing the batch fermentation of recombinant E. coli KO11 in the condensate. The model is based on Monod kinetics including substrate and product inhibition and the sum of the inhibition exerted by the rest of the inhibitors, lumped together. (C) 1995 John Wiley and Sons, Inc.
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| 2. |
- Triantafyllou, Angeliki Öste, et al.
(författare)
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Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media
- 1995
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 45:5, s. 406-414
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Tidskriftsartikel (refereegranskat)abstract
- The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (aw = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol‐containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N‐acetyl‐L‐phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol‐containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin–sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89°C). The transition temperatures of the substrate‐containing chymotrypsin and of the control were identical (72°C). © 1995 John Wiley & Sons, Inc.
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| 3. |
- Frenander, U, et al.
(författare)
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Cell harvesting by cross-flow microfiltration using a shear-enhanced module
- 1996
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 52:3, s. 397-403
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Tidskriftsartikel (refereegranskat)abstract
- Protein, produced by a bacterial culture of recombinant Vibrio cholerae, was separated from cells in a fermentation broth by cross-flow microfiltration. A new, mechanically agitated (rotational) shear filter, the DMF(TM) filter from Pall, was used to perform the separation. Higher protein recovery and permeate flux than commonly obtained during cell harvesting were demonstrated using sixfold concentration followed by twofold diafiltration. The transmembrane pressure only increased by 10 kPa when the flux was kept constant at 150 L/m(2) h during both concentration and diafiltration. The protein transmission was about 100% initially, and over 90% at the end of the concentration process. The protein transmission during the diafiltration was over 80%. The total recovery of protein was 97%. When using an enzymatic cleaning agent, no significant pure water flux decrease was detected during the course of the experiments. (C) 1996 John Wiley & Sons, Inc.
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| 4. |
- Medve, József, et al.
(författare)
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Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma reesei: Adsorption, sugar production pattern, and synergism of the enzymes
- 1998
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 59:5, s. 621-634
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Tidskriftsartikel (refereegranskat)abstract
- Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40degreesC) and enzyme loading (0.16 µmol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 µmol/g at 4degreesC and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60-90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40degreesC when the enzymes were added together they produced 27-45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4degreesC, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11-15%, 82-89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:621-634, 1998.
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| 5. |
- Planas, Jordi, et al.
(författare)
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Analysis of phase composition in aqueous two-phase systems using a two-column chromatographic method: Application to lactic acid production by extractive fermentation
- 1997
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 54:4, s. 303-311
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Tidskriftsartikel (refereegranskat)abstract
- A new chromatographic system for the simultaneous analysis of polyethylene glycol, dextran, sugars, and low-molecular-weight fatty acids was developed. The system is based on a gel exclusion column which allows a first separation between high- and low-molecular-weight compounds, and a cationic exchange column used to further separate the low-molecular-weight compounds. Two applications of the system were demonstrated: (i) after optimizing eluent conditions the gel exclusion column was used to determine the influence of lactic acid, phosphate buffer, and lactic acid bacteria on the ethylene oxide propylene oxide-dextran T40 phase diagram by HPLC; (ii) the ion exchange column was coupled in series with the gel exclusion column and the concentration of polyethylene glycol, dextran, glucose, lactate, acetate, and formate was determined in samples from the fermentative production of lactic acid in a polyethylene glycol 8000-dextran T40 aqueous two-phase system. The fermentation was operated without pH control in a repeated extractive batch mode, where the cell-free top phase was replaced four times, whereas the cell-containing bottom phase was reused repeatedly. The yield was 1.1 mol of lactic acid formed per mole of glucose added and the productivity was 4.7 mM.h-1. The polymeric composition of the fermentation system was monitored during the five repeated extractive batches, and it showed a progressive depletion in polyethylene glycol and a progressive enrichment in dextran. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 303-311, 1997.
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| 6. |
- Planas, Jordi, et al.
(författare)
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Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction
- 1999
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 66:4, s. 211-218
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Tidskriftsartikel (refereegranskat)abstract
- A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35degreesC at 5% phosphate, and increased linearly to 63degreesC at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 211-218, 1999.
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| 7. |
- Senthuran, A, et al.
(författare)
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Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei.
- 1997
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 55:6, s. 841-853
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Tidskriftsartikel (refereegranskat)abstract
- Lactic acid production by recycle batch fermentation using immobilized cells of Lactobacillus casei subsp. rhamnosus was studied. The culture medium was composed of whey treated with an endoprotease, and supplemented with 2.5 g/L of yeast extract and 0.18 mM Mn(2+) ions. The fermentation set-up comprised of a column packed with polyethyleneimine-coated foam glass particles, Pora-bact A, and connected with recirculation to a stirred tank reactor vessel for pH control. The immobilization of L. casei was performed simply by circulating the culture medium inoculated with the organism over the beads. At this stage, a long lag period preceded the cell growth and lactic acid production. Subsequently, for recycle batch fermentations using the immobilized cells, the reducing sugar concentration of the medium was increased to 100 g/L by addition of glucose. The lactic acid production started immediately after onset of fermentation and the average reactor productivity during repeated cycles was about 4.3 to 4.6 g/L . h, with complete substrate utilization and more than 90% product yield. Sugar consumption and lactate yield were maintained at the same level with increase in medium volume up to at least 10 times that of the immobilized biocatalyst. The liberation of significant amounts of cells into the medium limited the number of fermentation cycles possible in a recycle batch mode. Use of lower yeast extract concentration reduced the amount of suspended biomass without significant change in productivity, thereby also increasing the number of fermentation cycles, and even maintained the D-lactate amount at low levels. The product was recovered from the clarified and decolorized broth by ion-exchange adsorption. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:841-853, 1997.
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| 8. |
- Torto, N, et al.
(författare)
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Monitoring of enzymatic hydrolysis of starch by microdialysis sampling coupled on-line to anion exchange chromatography and integrated pulsed electrochemical detection using post-column switching
- 1997
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 56:5, s. 546-554
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Tidskriftsartikel (refereegranskat)abstract
- A quantitative evaluation of the hydrolysis of wheat starch using Termamyl, a thermostable alpha-amylase (endo-l,4-alpha-D-glucan, glucanohydrolase; EC 3.2.1.78), is reported. Data from the monitoring of the hydrolysis of wheat starch indicated that, after 1 h, glucose and maltooligosaccharides up to DP 7 were the main hydrolysis products and thus enabled optimization of a liquefication step during the production of L-lactic acid. The monitoring system used, both in the on- and off-line mode, was based on continuous flow microdialysis sampling (CFMS) coupled to anion exchange chromatography and integrated pulsed electrochemical detection (IPED). A microdialysis probe equipped with a 5-mm polysulfone (SPS 4005) membrane, with a molecular-weight cut-off of 5 kDa, was used to sample the hydrolysis products of native wheat starch at 90 degrees C. Characteristic fingerprint separations were achieved by anion exchange chromatography after enzymatic hydrolysis. Post-column switching improved the detection and, consequently, also quantification of the hydrolysates as fouling of the electrode could be reduced. Maltooligosaccharide standards were used for quantification and to verify the elution of the hydrolysates by spiking the off-line samples. (C) 1997 John Wiley & Sons, Inc.
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| 9. |
- Wehtje, Ernst, et al.
(författare)
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Water activity and substrate concentration effects on lipase activity.
- 1997
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 55:5, s. 798-806
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Tidskriftsartikel (refereegranskat)abstract
- Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.
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| 10. |
- Åkesson, Mats, et al.
(författare)
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On-line Detection of Acetate Formation in Escherichia coli Cultures using Dissolved Oxygen Responses to Feed Transients
- 1999
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Ingår i: Biotechnology and Bioengineering. - 1097-0290. ; 64:5, s. 590-598
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding.
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