bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of medium carbon‐to‐nitrogen ratio on biofilm formation and plasmid stability
Authors: Huang Ching‐Tsan , Peretti Steven W. , Bryers James D.
Published: December 2004

Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5α (pMJR1750) were investigated under different medium‐carbon‐to‐nitrogen (C/N) ratios. At C/N ratios of 0.07 and 1, net accumulation of both biofilm plasmid‐bearing and plasmid‐free cells continued through the entire experiment without attaining any apparent steady state. At C/N ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At C/N ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2g alginate equivalent/cm2 by the end of both experiments. At a C/N ratio of 5, polysaccharide increase significantly after 84 h, reaching about 7μg alginate equivalent/cm2 prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 μg alginate equivalent/cm2 at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 μg/cm2, respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 μg/cm2, respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 205 μg polysaccharide/μg protein, whereas those at C/N ratios of 5 and 10 increased to about 7 and 12 μg polysaccharide/μg protein, respectively.

Probabilities of plasmid loss in the biofilm cultures increased with increasing C/N ratios. At C/N ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.0013 ± 0.011, 0.020 ± 0.006 and 0.122 ± 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.38 ± 0.125. The increase of probability of plasmid loss at higher C/N ratios results from competition between cell replication and extracellular polysaccharide production. © 1994 John Wiley & Sons, Inc.
Mass transfer limitation of sulfate in methanogenic aggregates
Authors: Overmeire Ann , Lens Piet , Verstraete Willy
Published: December 2004

The role of mass transfer limitation of sulfate as a factor governing the competition between sulfate reducing and methane producing bacteria in methanogenic aggregates was theoretically evaluated by the calculation of steady‐state sulfate microprofiles using a reference set of parameters obtained from the literature. The shooting method was used as a numerical technique for solving the mathematical model. The effect of the parameters on mass transport limitation was tested by varying each reference value of the parameters with a factor of 3. Sulfate limitation within granules prevailed at moderate (0.1 kg m−3) and low sulfate concentrations in the bulk liquid, at high maximum sulfate utilization rates (3.73 × 10−5 kg SO kg−1 VSS S−1 or biomass concentrations (40 KG VSS m−3), and in large aggregates (radius of 7.5 10−4 m). The effective diffusion coefficient of sulfate and the affinity constant were less determinative for the penetration depth of sulfate within a granule. © 1994 John Wiley & Sons, Inc.
Novel redox surfactants and their interactions with glucose oxidase of Aspergillus niger
Authors: Saudan Pascale , Zakeeruddin Shaik Mohammed , Malavallon Muriell‐Anne , Grätzel Michael , Fraser David Michael
Published: December 2004

A number of novel redox surfactants (based on mixed bipyridine/dipyridylamine complexes of osmium (II) where the dipyridylamine ligands bears a saturated C8, C10, C12, C14, or C16 alkyl chain) were synthesized and characterized electrochemically and biochemically as mediators for glucose oxidase (EC 1.1.3.4, GOD) of Aspergillus niger. These compounds exhibited critical micelle concentrations (CMCs) in phosphate‐buffered saline solution (pH 7.4) in the range 10−4 10 10−3 M, the value decreasing with increasing chain length. Dependence of a number of properties (speed of mediation, redox potential, denaturing action on the enzyme, adsorption on an electrode surface) on the length of the mediator alkyl chain was observed. The presence of an alkyl chain decreased the rate of mediation relative to otherwise similar nonsurfactant mediators, and the longer alkyl chain, the slower the rate of mediation. For each compound, mediation above the CMC was about tenfold slower than that observed below the CMC. However, for the cases of mediator absorbed on an electrode surface with GOD, longer chains give increased physisorption of mixed micelles of enzyme and mediator. The compounds were incidentally found to inhibit the glucose oxidase activity of GOD in a complex manner; inhibition increased with increasing chain length and the deactivation, for any given compound, was more pronounced below the CMC than above. Glucose oxidase activity assays and study of the action of surfactants and mediators on the fluorescent properties of carboxy‐fluorescein‐labeled GOD led to the consideration of a model for redox surfactant‐GOD interaction where three mechanisms may operate: first, a selective interaction of mediators with the GOD active site; second, a nondenaturing association of short‐chain (<C14) cationic surfactants; and third, a denaturing action of long‐chain (⪆C14) cationic surfactants. © 1994 John Wiley & Sons, Inc.
Metabolic monitoring by using the rate of change of NAD(P)H fluorescene
Authors: Kwong Simon C. W. , Rao Govind
Published: December 2004

The amino acid fermentation by Corynebacterium glutamicum was monitored with an new technique that uses the first derivative of the NAD(P)H fluorescene signal. The rate of change of NAD(P)H pools is indicative of intracellular redox balance variations that correspond to metabolic changes. The profile of this signal showed several characteristics that coincided with major metabolic events during fermentation. We show here that the derivative fluorescence signal can accurately estimate points of threonine depletion, viable cell count, and the end of amino acid formation. Furthermore, on‐line optimization strategies can be developed by using the derivative fluorescene signal. © 1994 John Wiley & Sons, Inc.
Examination of primary metabolic pathways in a murine hybridoma with carbon‐13 nuclear magnetic resonance spectroscopy
Authors: Mancuso Anthony , Sharfstein Susan T. , Tucker Sean N. , Clark Douglas S. , Blanch Harvey W.
Published: December 2004

Primary metabolism of a murine hybridoma was probed with 13C nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1−13C] glucose, [2−13C] glucose, and [3−13C] glutamine. In vivo spectroscopy of the culture was used in conjunction with off‐line spectroscopy of the medium to determine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to levels observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/melate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; This value is a lower limit and the actual value may be higher. Incorporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal to the rate of glutamine uptake. The rate of incorporation of glucosederived acetyl‐CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and α‐ketoglutarate was 110% of the glutamine uptake rate. © 1994 John Wiley & Sons, Inc.
Biological sulphate reduction using gas‐lift reactors fed with hydrogen and carbon dioxide as energy and carbon source
Authors: van Houten Renze T. , Pol Look W. Hulshoff , Lettinga Gatze
Published: December 2004

Feasibility and engineering aspects of biological sulphate reduction in gas‐lift reactors were studied. Hydrogen and carbon dioxide were used as energy and carbon source. Attention was paid to biofilm formation, sulphide toxicity, sulphate conversion rate optimization, and gasliquid mass transfer limitations. Sulphate‐reducing bacteria formed stable biofilms on pumice particles. Biofilm formation was not observed when basalt particles were used. However, use of basalt particles led to the formation of granules of sulphate‐reducing biomass. The sulphate‐reducing bacteria, grown on pumice, easily adapted to free H2S concentrations up to 450 mg/L. Biofilm growth rate then equilibrated biomass loss rate. These high free H2S concentrations caused reversible inhibition rather than acute toxicity. When free H2S concentrations were kept below 450 mg/L, a maximum sulphate conversion rate of 30 g SO42−/L · d could be achieved after only 10 days of operation. Gas‐to‐liquid hydrogen mass transfer capacity of the reactor determined the maximum sulphate conversion rate. © 1994 John Wiley & Sons, Inc.
Protein extraction by reverse micelles: Studies on the recovery of horseradish peroxidase
Authors: Regalado Carlos , Asenjo Juan A. , Pyle D. Leo
Published: December 2004

Phase transfer studies were carried out on the solubilization of horseradish peroxidase (HRP) (E.C. 1.11.1.7) in reverse micelles formed in isooctane using the anionic surfactant, aerosol OT, at concentrations between 50 and 110mM. The selectivity of this methodology was tested, because the HRP used comprised a mixture of seven different isoenzymes with a wide range of isoelectric points. Forward and backward transfers were carried out in wellstirred vessels until equilibrium was reached. Significant protein partitioning could only be obtained by using NaCl to adjust ionic strength in pH range between 1.5 and 3.5, with a maximum at pH 3. The back transfer process was best at pH 8 with 80mM phosphate buffer and 1 M KCI. A loss of 1% to 3% of the surfactant through precipitation at the interface at pH<4 was observed, which may be due to instability in this pH region, because, even without protein, a similar precipitate was noticed. Protein partitioning was approximately constant when the ionic strength was increased up to 1 MNaCl at pH 3, but protein recovery in back transfer decreased accordingly. Hydrophobic interactions together with association between the protein and surfactant might be responsible for that behavior. Protein partitioning remained the same when the surfactant concentration was decreased to 50 mM, at the expense of higher variability. HPLC chromatograms showed no apparent damage to the protein after reverse micellar extraction. Protein partitioning is best when the temperature is kept at 25×C. The amount of protein and specific activity recovered strongly depends on the phase ratio used during forward transfer. Overall activity recovery varied from 87% to 136% when the phase ratio was increased from 1:1 to 30:1 in forward transfer. This behavior may be due to a change in the ratio of the three isoenzymes recovered after the backward transfer process, with the most active one being increasingly enriched at higher phase ratios. © 1994 John Wiley & Sons, Inc.
Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures
Authors: Ljunggren Jan , Häggström Lene
Published: December 2004

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures μ reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported Ks vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual‐substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NHx/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual‐substrate‐limited fed batch culture as judged by a comparison of μ in the different cultures. © 1994 John Wiley & Sons, Inc.
Efficient Hydrogen photoproduction by synchronously grown cells of a marine cyanobacterium, Synechococcus sp. Miami BG 043511, under high cell density conditions
Authors: Kumazawa Shuzo , Mitsui Akira
Published: December 2004

The capability of hydrogen photoproduction under high cell density conditions was examined using synchronously grown cells of nitrogen‐fixing Synechococcus sp. Miami BG 043511. Optimum hydrogen yield was obtained when vessels (25 ml) contained 0.2 to 0.3 mg chlorophyll a in 3‐mL cell suspension. During a 24‐h incubation period, an initial phase of hydrogen and carbon dioxide production and a subsequent phase of carbon dioxide uptake and oxygen accumulated as major products after 24 h. after the initial 24‐h. After the initial 24‐h incubation, as high as 7.4 and 3.7 L (at standard condition) of hydrogen and oxygen, respectively, accumulated in vessels with 22‐ml gas phase. This indicated that the pressure in the flask increased to 1.5 atmosphere. Energy conversion efficiency based on photosynthetically active radiation (25 W/m2) was about 2.6%. However, increased pressure somehow reduced the duration of hydrogen production. Duration of hydrogen and oxygen production was prolonged by periodical (24‐h interval) gas replacement during incubation. © 1994 John Wiley & Sons, Inc.
Formation of carotenoids by rhodotorula glutinis in whey ultrafiltrate
Authors: Frengova Ginka , Simova Emilina , Pavlova Konstantza , Beshkova Dora , Grigorova Drinka
Published: December 2004

The growth and carotenoid biosynthesis of the yeast Rhodotorula glutinis was studied by cocultivation with Lactobacillus helveticus in cheese ultrafiltrate containing 3.9% and 7.1% lactose. By growing this mixed culture in a 15‐L fermentor MBR AG (Switzerland) at an air flow rate of 0.5 L/L min and agitation at 220 rpm for 6 days, a total yield of carotenoids of 268 μg/g dry cells wasobtained. Carotenoids were formed almost parallel with the cell growth, anda maximum production was reached at an early stationary phase. A high‐performance liquid chromatographic system (HPLC) permitting simultaneous determination of major carotenoid pigments was used. The three main pigments (torularhodin, β‐carotene, and torulene) were formed in Rhodotorula glutinis, and reached a maximum concentration as follows: 182.0, 43.9, 23.0 μg,g dry cells. © 1994 John Wiley & Sons, Inc.
Efficient specific release of periplasmic proteins from Escherichia coli using temperature induction of cloned kil gene of pMB9
Authors: Steidler Lothar , Fiers Walter , Remaut Erik
Published: December 2004

We have cloned the kil gene of pMB9 under control of the tightly regulated leftward promoter (pL) of coliphage λ. Three types of plasmids were constructed. In all cases the activity of the λ promoter is controlled by a thermosensitive cl repressor (product of the c/857 gene) supplied form a resident defective prophage or cloned onto a compatible p 15A‐derived plasmid. Induction of the kil protein is brought about by a temperature shift of the culture from 28°C to 42°C. Plasmid pPLc28K1 contains the kil gene including its natural ribosome‐binding site and preceded by a transcription termination site. Using a bacterial strain with antitermination properties (e.g., M5219), periplasmic proteins can upon induction be gradually the growth of the host strain. The second plasmid pPLc321K1, contains the kil‐coding sequence preceded by an engineered ribosome binding site derived from the attenuator of the Escherichia coli tryptophan operon. With this plasmid induction of the Kil protein is very rapid and specific release of the periplasmic proteins in essentially complete within 30 min after induction. In a third construct, pcl857K1, the pL‐kil cassette together with c/857 allele are present on the same replicon, which is compatible with ColE1‐derived expression vectors. This configuration allows accumulation in the periplasm of cloned gene products, induced by, e.g., tac or trp promoters at low temperature and subsequent release into the medium following increase of the temperature of the culture. Under repressed conditions (growth at low temperature) all plasmids are perfectly stable in a large number of E. coli strains tested, also when cultivated on a 20‐L fermentor scale. Controlled, heat‐induced release of periplasmic proteins is highly specific and applicable at relatively high cell densities. The method therefore is an attractive alternative to cumbersome osmotic shock procedures for large‐scale cultures. © 1994 John Wiley & Sons, Inc.
Saccharification, fermentation, and protein recovery from low‐temperature AFEX‐treated coastal bermudagrass
Authors: Holtzapple Mark T. , Ripley E. Payson , Nikolaou Michael
Published: December 2004

Coastal bermudagrass was pretreated by a low‐temperature ammonia fiber explosion (AFEX) process, which soaked the grass in liquid ammonia and then explosively released the pressure. Saccharifying enzymes were systematically applied to the AFEX‐treated grass corresponding to low, medium, and high loadings of cellulase/hemicellulase (from Trichoderma reesei), cellobiase, glucoamylase, and pectinase. Three‐day sugar yields linearly correlated with the logarithm of the cellulase loading. Supplemental enzymes (cellobiase, pectinase) caused upward shifts in the lines. The linearity and upward shifts are consistent with the HCH‐1 model of cellulose hydrolysis. The hydrolysis sugars were converted to ethanol using yeast (Saccharomyces cerevisiae). The solid residues were treated with proteases to attempt recovery of valuable proteins. The low‐temperature AFEX pretreatment was able o nearly double sugar yields. At the highest cellulase loadings (30 IU/g), the best reducing sugar and ethanol yields were 53% and 44% of the maximum potential, respectively. Protein recovery was, at most, 59% © 1994 John Wiley & Sons, Inc.
Broth recycle in a yeast fermentation
Authors: Hsiao Tzu‐Yin , Glatz Charles E. , Glatz Bonita A.
Published: December 2004

Fermentation is a water‐intensive process requiring treatment of large amounts of effluent broth. It is desirable to increase the ratio of product produced to the volume of effluent by minimizing the discharge of effluent from the fermentation process. A study of recycling spent fermentation process. A study of recycling spent fermentation broth for the subsequent fermentation was carried out with Apiotrichum curvatum an oleaginous yeast, as the working culture. Spent broth from a defined medium was recycled t replace as much as 75% of the water and salts for subsequent batches and this was repeated for seven sequential batches without affecting cell mass and lipid production. A 64% vlume reduction of wastewater was achieved in this manner. However, when using whey permeate as the medium, lipid production dropped after three consecutive recycle operations at 50% recycle, and after two consecutive recycle operations at 75% and 100% recycle. Accumulation of ions in the broth appeared to be responsible for the inhibition. An ion exchange step was able to eliminate the ion buildup and restore fermentation performance. © 1994 John Wiley & Sons, Inc.
The existence of a biological equilibrium in a trickling filter for waste gas purification
Authors: Diks R. M. M. , Ottengraf S. P. P. , Vrijlnad S.
Published: December 2004

Clogging is well‐known phenomenon in the application of a biological tricking filter for both waste gas and wastewater treatment. Nevertheless, no such observations or even significant changes in pressure drop have ever been recorded during the long‐term processing of a waste gas containing dichloromethane (DCM) as a sole carbon source. To obtain more information about this phenomenon, a detailed investigation into the carbon balance of this system has been performed. During a period of operation of about 200 days the rate of DCM elimination and the overall rate of CO2 production in a continuously operating filter were therefore recorded daily, thus allowing an evaluation of the overall conversion process. Furthermore pseudo‐steady‐state measurements were carried out on a regular basis. These experiments reveal more detailed information on the actual DCM conversion by Hyphomicrobium GJ21 within the biofilm. The combined results of the experiments described in this article show that on an overall basis a so‐called biological equilibrium, i.e., a situation of no net biomass accumulation, is obtained in the course of time. It appeared that the overall rate of CO2 production slowly increased until, after some 200 days, it finally counter‐balanced the conversion rate of DCM on a molar‐basis. As opposed to this result, all pseudo‐steady‐state experiments indicated that about 60% of the eliminated primary carbon source is converted into biomass. This is in good agreements with results from microkinetic experiments. Based on these results and evaluation of the experimental data, it is concluded that interactions between several microbial populations are involved in this biological equilibrium. These interactions include both biomass growth and biomass degradation. © 1994 John Wiley & Sons, Inc.
An Escherichia coli plasmid vector system for production of streptavidin fusion proteins: Expression and bioselective adsorption of streptavidin‐β‐galactosidase
Authors: Walsh Marie K. , Swaisgood Harold E.
Published: December 2004

Covalently immobilized biotin was used as a biospecific adsorbant to investigate the application of streptavidin as an affinity domain for simultaneous purification and immobilization of recombinant proteins. A streptavidin‐β‐galactosidase fusion protein was constructed and tested as a model system. The gene for streptavidin from Streptomyces avidinii was modified by polymerase chain reaction to mutate the stop codon and to facilitate cloning into an Escherichia coli expression vector yielding a versatile plasmid with 37 unique restriction enzyme sites at the 3' end. E. coli β‐galactosidase was cloned in‐frame to the streptavidin gene. Analysis of lysates of induced recombinant E. coli cells by SDS‐PAGE and Western blots indicated that the 133.6‐kDa fusion protein was expressed. Sulfosuccinimidyl‐6‐(biotinamido) hexanoate was covalently immobilized on 3‐aminopropyl‐controled‐pore glass beads. Exposure of recombinant cell lysates to this support indicated that streptavidin‐β‐galactosidase was bioselectively adsorbed. The resulting biocatalyst contained 300 mg protein per gram of beads and exhibited a specific activity of 306 βmol/min per milligram protein with o‐nitrophenyl‐β‐D‐galactopyranoside as substrate corresponding to approximately 50% of that observed for commercially pure E. coli β‐galactosidase. © 1994 John Wiley & Sons, Inc.
Synthesis of protein‐containing polymers in organic solvents
Authors: Yang Zhen , Williams Darrel , Russell Alan J.
Published: December 2004

Subtilisin has been modified with polyethylene glycol (PEG) monomethacrylate (MW 8000) by reductive alkylation, and incorporated into polymethyl methacrylate durring free‐radical initiated polymerization. The activity and stability of the PEG‐modified enzymes have been determined in aqueous buffer and organic solvents. The Km and Vmax values for unmodified, singly and doubly modified subtilisin were compared in these environments, and the half‐lives of both modified enzymes were remarkably high (up to 2 months). The protein‐containing polymer was analyzed for activity and polymer properties, and our results indicate that active subtilisin can be incorporated into polymethyl methacrylate during polymerization in organic solvents while retaining its activity and stability. © 1995 John Wiley & Sons, Inc.
On‐line gas analysis in animal cell cultivation: II. Methods for oxygen uptake rate estimation and its application to controlled feeding of glutamine
Authors: Eyer K. , Oeggerli A. , Heinzle E.
Published: December 2004

Different methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge of KLa, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, a new method of aeration was designed. Oxygen and CO2 transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100‐fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O2, CO2, Ar, and N2. The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on‐line methods for OUR measurement were tested during the cultivation of animal cells. The new method allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. © 1995 John Wiley & Sons, Inc.
Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media
Authors: Triantafyllou Angeliki Öste , Wehtje Ernst , Adlercreutz Patrick , Mattiasson Bo
Published: December 2004

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.
Properties of microfiltration membranes: Mechanisms of flux loss in the recovery of an enzyme
Authors: Bowen W. Richard , Hall Nicola J.
Published: December 2004

The transmission and rate of filtration of the enzyme yeast alcohol dehydrogenase (YADH) has been studied at capillary pore microfiltration membranes. Photon correlation spectroscopy (PCS) with nanometer resolution showed that the enzyme existed as discreate molecules only for a narrow range of pH and ionic strength. Under such conditions, the transmission of the enzyme was high. However, the rate of filtration still decreased continuously with time. Analyssis of the time dependence of the rate of filtration indicated that this decrease was due to in‐pore enzyme deposition at low concentration (“standard blocking model”) and suface depositon at high concentration (“cake filtration model”). Use of atomic force microscopy (AFM) gave unequivocal and quantitative confirmation of these inferences. The work shows the great advantage of using advanced physical characterization techniques, both for the identification of the optimum conditions for filtration (PCS) and for the elucidation of mechanisms giving rise to inefficiencies in the filtration process (AFM). © 1995 John Wiley & Sons, Inc.
Genetically engineered charge modifications to enhance protein separation in aqueous two‐phase systems: Charge directed partitioning
Authors: Luther John R. , Glatz Charles E.
Published: December 2004

This report continues or examination of the effect of genetically engineered charge modifications on the partitioning behavior of proteins in aqueous two‐phase extration. The genetic modifications consisted of the fusion of charged peptide tails to β‐galactosidase and charge‐change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a function of interfacial potential difference. In this study, we examined charge directed partitioning behavior in PEG/dextran systems containing small amounts of the charged polymers diethylaminoethyl‐dextran (DEAE‐dextran) or dextran sulfate. The best results were obtained when attractive forces between the protein and polymer were present. Nearly 100% of the β‐galactosidase, which carries a net negative charge, partitioned to the DEAE‐dextran‐rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein‐rich phases suggest that strong charge interactions resulted in protein/polymer aggregation, which may have contributed to the extreme partitioning. Unlike the potentialdriven partitioning reported previously, consistent partitioning trends were observed as a result of the fusion tails, with observed shifts in partition coefficient (Kp) of up to 37‐fold. However, these changes could not be solely attributed to charge‐based interactions. Similarly, T4 lysozyme, carrying a net positive charge, partitioned to the dextran sulfate‐containing phase, and displayed four‐ to sevenfold shifts in Kp as a result of the point mutations. These shifts were two to four times stronger than those observed for potential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of β‐galactosidase with polyarginine tails. The high positive charge density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2‐ to 15‐fold shifts in Kp. © 1995 John Wiley & Sons, Inc.
Factors affecting the esterification of lauric acid using an immobilized biocatalyst: Enzyme characterization and studies in a well‐mixed reactor
Authors: Lima F. Vázquez , Pyle D. L. , Asenjo J. A.
Published: December 2004

The esterification of lauric acid with geraniol catalyzed by the commercially immobilized lipase preparation from Mucor miehei, Lipozyme®, was studied in well‐stirred flasks. The enzyme support was characterized in terms of its internal and external surface area, protein location, and protein content. It was found that the enzyme was mainly located on the external surface of the support, therefore, internal diffusional limitations were not important. It was also shown that the protein content of the support depends on the size of the particle, with smaller particles containing higher amounts of protein per unit weight. Under the conditions studied, the reaction was not under external mass transfer limitations, and the initial reaction rate depended on the size of the support particles. This was mainly due to the different protein contents on the support as a function of particle size and not to internal or external mass transfer limitations. Also, it was found that the inhibition exerted by water was predominantly a physical effect due to its accumulation around the enzyme. It was also found that the reaction was substrate inhibited by lauric acid, but not by geraniol. © 1995 John Wiley & Sons, Inc.
Determination of the volumetric mass transfer coefficient (kLa) using the dynamic “gas out–gas in” method: Analysis of errors caused by dissolved oxygen probes
Authors: Tribe L. A. , Briens C. L. , Margaritis A.
Published: December 2004

There are many dynamic methods for measuring the volumetric mass transfer coefficient. The “gas out–gas in” method can directly determine the volumetric mass transfer coefficient in a bioreactor system and provide estimates of the volumetric microbial oxygen uptake rate and the average oxygen saturation concentration at the gas–liquid interface. The errors on these parameters are large if the dissolved oxygen probe response time is not considered. For reliable measurements, deconvolution of the oxygen probe measurements must be made. © 1995 John Wiley & Sons, Inc.
Intracellular responses of productive hybridomas subjected to high osmotic pressure
Authors: Oh Steve K. W. , Chua Florence K. F. , Choo Andre B. H.
Published: December 2004

It has previously been found tht hybridoma cells undr hyuerosmotic stress produce higher amounts of antibody. This study indentified the cellular processes and mechanisms that occur during this event. In studies fo hybridomas adpated toosmolarities ranging between 300 and 450 mOsm (uusing NaCl), antibody production increased to a saturation level while cell growth decreased progressively. At 500 mOsm, lower, cell numbers and markedly decreaased productivity resulted. Sucrose and KCl were found to induce similar trends, except to different extents.

Several important change in cellulaes in cellular responses were onsserved. Elevation of osmnolarity with NaCl from 300 to 350 mOsm causes an increase of zwiterionic amino acid upatake, which, occurredvia Na+‐dependent transport systems. In particuar, systedm A was enhanced by 1.86‐fold, but noenhancement was observed for Na+‐independent transport systems, In addition, amino acids reactive with Na+‐dependent transport systems were onserved to be abundant within osmotically stressed hybridomas in the middle and dlate exponentoial statges. Sucroses ans Kcl caused similar uptake effects, but to a laeeser degree, as long as sodium ions were present in solution.

Specific consumption rates fo glucose and glutamine incresase by 19% and 20%, respectively, under high osmolarity treatment. Thewse increases were confirmed by the 5% to 10% increase in cellular metabolic acitivity. At 350 mOsm, growth rate was slower, compared with the 300‐mOsm culture, which was reflected by thelower DNA conetr4ation. Stressed cultures contained enhanced leyls of tatal RNA content could in turn increase the translation rates of proteins. This was reflected in the accumulation of both dry cell weight and total cellular protein at linear rates of 0.42 μG/106 cells/mOsm and 0.21 μg/106 cells/mOmsm, respectively, with increasing osmolarty between 300 and 450 mOsm.

Overall, hybridoms increased their metabolic activities and amino acids uptake via the Na+‐dependent symports to compensate for teh osmotically elevated external environment. These effects contribute directly and indirectly tothe increased cell mass consisting of a larger pool of amono acids, RNA, cellular proteins, and seecreted antibody produt. © 1995 John Wiley & Sons, Inc.
Physico‐chemical properties of the encapsulation matrix and germination of carrot somatic embryos
Authors: Timbert Raphaëlle , Barbotin Jean‐Noël , Kersulec Alain , Bazinet Christophe , Thomas Daniel
Published: December 2004

Carrot somatic embryos were encapsulated in alginate gel beads. To improve the quality of a “synthetic seed” coating, the rheology and dehydration properties of different matrices were tested. By increasing alginate and CaCl2 concentrations, additional mineral elements were shown to increase resistance to rupture, and to depress the germination of somatic embryos. A polysaccharide addition was found to slow the alginate matrix dehydration; alginate‐gellan gum and alginate‐kaolin matrices could preserve the viability of somatic embryos at low relative humidities (30% to 35% germinations at 50% relative humidity) to a greater extent than other matrices. © 1995 John Wiley & Sons, Inc.
High viable cell concentration fed‐batch cultures of hybridoma cells through on‐line nutrient feeding
Authors: Zhou Weichang , Rehm Jutta , Hu Wei‐Shou
Published: December 2004

A hybridoma cell line was cultivated in fed‐batch cultures using a low‐protein, serum‐free medium. On‐line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on‐line using the stoichiometric relations between glucose and oxygen consumption. Through on‐line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 × 106 cells/mL, a very high concentration of 1.36 × 107 cells/mL with a high cell viability (>90%) was achieved in the fed‐batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well‐closed material balance was established. Our results demonstrate the value of coupling on‐line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. © 1995 John Wiley & Sons, Inc.
Preparation and in vitro analysis of microencapsulated genetically engineered E. coli DH5 cells for urea and ammonia removal
Authors: Prakash S. , Chang T. M. S.
Published: December 2004

This article describes a novel method of urea and ammonia removal using microencapsulated, genetically engineered Escherichia coli DH5 cells. Optimization of bacterial cell encapsulation was carried out. The optimal method consists of alginate 2.00% (w/v) at a flow rate of 0.0724 mL/min and a coaxial air flow rate of 2.00 L/min. This produces spherical, alginate‐poly‐L‐lysine‐alginate (APA) microcapsules of an average 500 ± 45 μm diameter. Increasing the concentration of alginate from 1.00% to 1.75% improves the quality of the microcapsules, while cell viability remains unaffected. The APA microcapsules are mechanically stable up to 210‐rpm agitation with no bacterial cell leakage. The in vitro performance of urea and ammonia removal by encapsulated bacteria is assessed. One hundred milligrams of bacterial cells in APA microcapsules, in their log phase state of growth, can lower 87.89 ± 2.25% of the plasma urea within 20 min and 99.99% in 30 min. The same amount of encapsulated bacteria can also lower ammonia from 975.14 ± 70.15 μM/L to 81.151 ± 7.37 μM/L in 30 min. There are no significant differences in depletion profiles by free and encapsulated bacteria for urea and ammonia removal. This novel approach using microencapsulated, genetically engineered E. coli cells is significantly more efficient than presently available methods of urea and ammonia removal. For instance, it is 30 times more efficient than the standard urease–ammonium adsorbent system. © 1995 John Wiley & Sons, Inc.
Growth kinetics of vitis vinifera cell suspension cultures: I. Shake flask cultures
Authors: Pépin Marie‐France , Archambault Jean , Chavarie Claude , Cormier François
Published: December 2004

Vitis vinifera cell suspension cultures carried out in shake flasks were closely examined for biomass growth and cell division in relation to carbohydrate, NH4, NO3PO4, and dissolved oxygen (DO)consumption. After inoculation, the oxygen uptake rate of the cultures measured on‐tine was observed to increase continuously to a maximum value of 3.8 mmol O2L−1h−1 at day 7 when cell division ceased and dissolved oxygen reached its lowest level of 17% air saturation. During this first phase of growth, the specific oxygen uptake rate remained constant at ∼0.6 mmol 02 O2 g−1 dw h−1or ∼2.2 μmol O2, (106 cells)−1 h−1 whereas dry biomass concentration increased exponentially from 1.5 to 6.0 g dw L−1. Thereafter, dry biomass concentration increased linearly to ∼14 g dw L−1 at day 14 following nitrate and carbohydrate uptake. During this second phase of growth, the biomass wet‐to‐dry weight ratio was found to increase in an inverse relationship with the estimated osmotic pressure of the culture medium. This corresponded to inflection points in the dry and wet biomass concentration and packed cell volume curves. Furthermore, growth and nutrient uptake results suggest that extracellular ammonium or phosphate ion availability may limit cell division. These findings indicate that cell division and biomass production of plant cell cultures may not always be completely associated, which suggests important new avenues to improve their productivity. © 1995 John Wiley & Sons, Inc.
Equilibria for the adsorption of antibiotics onto neutral polymeric sorbents: Experimental and modeling studies
Authors: Chaubal Mahesh V. , Payne Gregory F. , Reynolds Charles H. , Albright Robert L.
Published: December 2004

The objective of this work was to study the equilibria for adsorption of three antibiotics (penicillin V, tetracycline, and cephalosporin C) from water onto commercially available neutral polymeric sorbents. The pH was observed to be an important factor in adsorption as our results suggest that the neutral forms of penicillin V and cephalosporin C are preferentially adsorbed onto the neutral sorbents. Also, sorbent surface chemistry was observed to be important for adsorption, as the antibiotics adsorbed more favorably (both in terms of affinities and enthalpies) onto the aromatic sorbent as compared to the aliphatic ester sorbent. In addition to these thermodynamic measurements, molecular modeling studies and Monte Carlo simulations suggest that adsorption onto aromatic sorbents may involve specific interactions between the planar regions of the antibiotic molecules and the phenyl rings of the aromatic sorbent. The interaction energies predicted from Monte Carlo simulations were observed to provide qualitative agreement with experimentally determined adsorption affinities. © 1995 John Wiley & Sons, Inc.
Stability of transfectomas producing chimeric antibody against the pre‐S2 surface antigen of hepatitis B virus during a long‐term culture
Authors: Bae Sung Won , Hong Hyo Jeong , Lee Gyun Min
Published: December 2004

To design the scheme of large‐scale production of chimeric antibody for the postexposure prophylaxis of hepatitis B virus (HBV) infection, the stability of transfectomas (H69K‐1 and 6‐31) in regard to antibody production was examined during a long‐term, repeated fed‐batch culture without selection pressure using antibiotics. Although the H69K‐1 transfectoma was more stable than the 6‐31 transfectoma, both displayed gradual decreases in specific antibody productivity (qAb) for the first several weeks of cultivation. During this period, qAb was de‐creased by 40% to 50%. This loss of qAb was due mainly to the appearance of a nonproducing population (NP) of transfectoma, which was monitored throughout the culture by flow cytometry and the limiting dilution method. However, an NP did not overtake the culture and was balanced with a producing population (P) of transfectoma, resulting in stable antibody production. The subclones of NP obtained at the end of long‐term culture were further characterized by reverse transcription‐polymerase chain reaction assay of the heavy and light chain mRNA. All the subclones of NP derived from H69K‐1 transfectoma had only light chain mRNA. On the other hand, an NP in the 6‐31 transfectoma culture was heterogeneous. Some subclones of NP derived from 6‐31 transfectoma had only heavy chain mRNA and other subclones had only light chain mRNA. Taken together, the results obtained here suggest that selection pressure is necessary for a long‐term, continuous culture, because stable antibody production in a long‐term culture was achieved only after a significant loss of antibody productivity. Accordingly, a batch culture appears to be more appropriate for large‐scale chimeric antibody production without selection pressure. © 1995 John Wiley & Sons, Inc.