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Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol-based processes lead to large proportions of biotin-blocked binding sites of streptavidin due to biotin-supplemented media. Targeting these problems, this paper provides strategies for the methanol-free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin-blocked streptavidin. The optimized, easily scalable fed-batch process led to a tetrameric product concentration of up to 4.16 +/- 0.11 mu M of biotin-free streptavidin and a productivity of 57.8 nM h(-1) based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un-optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates ( approximate to D

Due to its various applications the protein streptavidin is a highly interesting target for heterologous production. This study focuses on different Escherichia coli-based constructs targeting a high-level expression and secretion of streptavidin to the medium. The effect of various promoters, variants of the target gene, leader sequences and host strains on expression and secretion into the culture broth was analyzed. Constitutive production of full-length streptavidin fused with the leader sequence of the bglA gene from Bacillus amyloliquefaciens by the periplasmic 'leaky mutant' E. coli JW1667-5 (Δlpp-752:kan) at 30°C generated the highest yield of the conditions tested, surpassing the extracellular concentration of a conventional T7-based expression system. Supplementation of the medium by the non-ionic surfactants Triton(®) X-100 and X-45 led to an improved secretion of the protein to the culture supernatant. Tetrameric concentrations of streptavidin of 2790±166nM were reached in shake flasks at a productivity of 49.6nMh(-1). Optimization of conditions led to a successful transfer to the bioreactor, yielding a maximal concentration of 2608±169nM and a productivity of 65.2nMh(-1) in fed-batch operation. The proportion of biotin-blocked binding sites of 8.3±4.3% indicated a highly bioactive product.

The protein streptavidin (SAV) is applied in a large variety of molecular methods due to an extraordinarily strong binding to the vitamin biotin (BIO). The protein structure is homotetrameric, characterized by one binding site for BIO per subunit. Therefore, one of the major criteria to determine the quality of SAV isolates is the proportion of BIO-blocked binding sites per tetramer. A rapid analysis of BIO-free binding sites is achieved by fluorescence quenching of biotin-4-fluorescein (B4F). However, BIO-blocked binding sites can only be determined by costly and laborious procedures such as ELISA-based methods or radioactive labeling. This study describes the systematic, model-supported development of a method for the quick and simple detection of BIO-blocked binding sites, based on a short-term heat incubation of the sample in the presence of B4F. Kinetic modeling and parameter estimation yielded dissociation constants of 1.22 +/- 0.27 x 10(-11) M for the complex SAV-BIO and 5.16 +/- 0.70 x 10(-13) M for SAV-B4F at 70 degrees C, allowing a displacement of SAV-bound BIO by B4F. This method allows the rapid monitoring of BIO-blocked binding sites in fermentation processes, independent from the chain length of SAV and the concentration of contaminating proteins, e.g. when optimizing the BIO concentration in cultivation media.

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200 h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.

Recently, it had been shown that Euglena gracilis was able to grow heterotrophically not only on synthetic media, but also on media based on potato liquor. Supplementation with glucose in both cases led to the accumulation of paramylon, a beta-1,3-glucan. Thus, such a process may yield a valuable product accompanied by the revaluation of an otherwise annoying waste stream of the potato-starch industry. Actually, process strategies have been evaluated in order to optimise the concentration of paramylon obtained at the end of the cultivation process. Therefore, cultivation processes based on fed-batch and in particular repeated-batch strategies have been studied. It is shown that repeated-batch operation maybe particularly suited for such a process since E. gracilis seems to adapt gradually to the cultivation medium so that the concentration of media components may be increased step by step. Repeated-batch cultivation of E. gracilis leads to biomass concentrations in access of 20 g/L with a consistent paramylon mass fraction of about 75%. Cultivations have been carried out at an operating temperature of 27.5 degrees C. As had been found earlier already, pH control is not required during cultivation. On the basis of these results it is clear that repeated-batch cultivation represent a simple and economic way for the production of paramylon by heterotrophic cultivation of E. gracilis.

In the present study, Dictyostelium discoideum AX2-R2 was grown on living bacteria Escherichia coli B/r in shake-flasks and in standard stirred tank bioreactors. The maximum cell densities of D. discoideum detected in shake-flasks and in bioreactors were almost identical with 1.3 x 10(7) and 1.4 x 10(7) ml(-1), respectively. More than 99% of the bacterial biomass was consumed in both cases. D. discoideum was cultivated in the presence of different porous supports with the aim of studying its growth in an immobilized state using living bacteria as substrate. For small scale, a shake-flask cultivation system with external loop was applied, while on a larger scale a bioreactor was coupled with an external loop of a glass column containing a fixed bed of the porous support. Under these conditions, the maximum cell density of immobilized D. discoideum cells was 5.8 x 10(7) ml(-1) on porous sintered glass (SIKUG 041) and 2.4 x 10(6) ml(-1) on silicone foam (ImmobaSil R), respectively. The success of the immobilization process was documented by scanning electron microscopy. (C) 2008 Elsevier Ltd. All rights reserved.

Die Verteilung von RNA und Plasmid-DNA wurde in einem inversmizellaren Zweiphasensystem bestehend aus Trioctylmethylammoniumchlorid (TOMAC) und Isooctan untersucht. Die Auswirkung verschiedener Salze und Alkohole auf den Transfer der Nukleinsauren wurde vor allem unter dem Gesichtspunkt einer hohen Wiederfindung der Plasmid-DNA nach der Ruckextraktion betrachtet. Systeme, die eine Trennung von Plasmid-DNA und RNA ermoglichen, wurden eingesetzt, um RNA aus bakteriellem Klarlysat zu entfernen.

Bei der Aufreinigung rekombinanter Proteine aus mikrobiellen Fermentationsprozessen stellt die extrazellulare Akkumulation des Produktes eine wesentliche Vereinfachung dar. Diese Arbeit zeigt, wie das Maltosebindeprotein (MBP), ein haufig eingesetzter Fusionspartner fur die Affinitatsreinigung, unter Coexpression von Bacteriocin freisetzenden Proteinen (BRP) auch eine Sekretion in das Kulturmedium ermoglicht. Dadurch wird die Gewinnung des Produktes deutlich erleichtert.

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals. Consequently, a treatment for the chemical deglycation with trifluoromethanesulfonic acid was applied, which resulted in the identification of protein Z4, LTP1, CMb, CMe, pUP13, 3a, and Bwiph as constituents of the haze proteome. Because only one hordein was detectable and the proline content in haze hydrolysates was lower than those of barley prolamins, our results suggest that this class of proteins is of minor importance for haze development.

The Gram-negative bacterium Escherichia coli B-3996 represents an interesting host organism for the production of the essential amino acid L-threonine. Microbial processes-especially those of aerobic cultivation-lead to the generation of considerable amounts of biomass, thus lowering the product yield. These are the reasons for studying methods for the recycling of biomass from E. coli. It will be shown that it is possible to disintegrate the microbial biomass-preferably by means of high pressure homogenisation followed by a protease treatment of the resulting slurry of debris-in an efficient way and to recycle at least different amounts of the soluble part as cultivation medium component. By studying the growth and product formation of E coli no adverse effects have been observed. (c) 2007 Elsevier B.V. All rights reserved.

We present a web-based integrated proteome database, termed 2DBase of Escherichia coli which was designed to store, compare, analyse, and retrieve various information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry. The main objectives of this database are (1) to provide the features for query and data-mining applications to access the stored proteomics data (2) to efficiently compare the specific protein spots present in the comparable proteome maps and (3) to analyse the data with the integrated classification for cellular functions of gene products of E. coli. This database currently contains 12 gels consisting of 1185 protein spots information in which 723 proteins were identified and annotated. Individual protein spots in the existing gels can be displayed, queried, analyzed, and compared in a tabular format based on various functional categories enabling quick and subsequent analyses. Our database satisfies the requirement to be a federated 2-DE database by accomplishing various tasks through a web interface providing access to a relational database system. The 2DBase of E. coli database can be accessed at http://2dbase.techfak.uni-bielefeld.de/.

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme. In this study we investigated the distribution of plasmid DNA and RNA in reverse micellar two-phase systems which is considered to be the basis for the development of an extractive purification step that can easily be integrated into common processes. For this purpose the distribution of the 4.6 kb plasmid pUT649 and Escherichia coli RNA in systems comprising isooctane, ethylhexanol, and the surfactant methyltrioctylammoniumchloride (TOMAC) under the influence of different salts was studied. Anion concentrations at which the partitioning behaviour for nucleic acids inverted (inversion point) were identified. Systems capable of separating RNA from plasmid DNA were further analysed and applied to extract RNA from plasmid DNA out of a preconditioned cleared lysate. The capability of reverse micellar systems for plasmid form separation was also shown by capillary and agarose gel electrophoresis. (C) 2007 Elsevier B.V. All rights reserved.

The main variable of enzymatic processes is often found to be the operating temperature. An increase in temperature leads to higher rates for the catalytic transformation. However, beyond a certain temperature catalyst deactivation is winning the game. Therefore, processes should be optimized in order to determine the temperature which leads to a minimal demand of enzyme preparation. For the prediction of such optimal reactor operation, modeling of the temperature dependence of the process has to be performed. Examples of such modeling are given for the hydrolysis of lactose in UHT milk by means of three different beta-galactosidases - those from Aspergillus orvzae, Kluvveromyces lactis, and Escherichia coli. The reaction kinetics for a constant initial lactose concentration can be described by a model of two parameters, of which only one depends on temperature. For the lactase of E coli the reaction can be described as a simple reaction with first order kinetics. The deactivation mechanism includes a reversible as well as an irreversible path of denaturation. The temperature dependent parameters follow Arrheilius' and van't Hoff's law, respectively. On the basis of their particular reaction models all three enzymes can be compared with respect to their Optimum use. The models have been verified under laboratory conditions and have shown their usefulness for the prediction of optimum operating variables. Quite remarkable features have been found for the lactase of E. coli.

Im Sinne eines produktintegrierten Umweltschutzes soll die Biomasse, die bei der Herstellung von biotechnischen Produkten auftreten kann, als wertvolle Nahrstoffquelle zur Schonung energetischer und stofflicher Ressourcen wieder in den Produktionsprozess zuruckgefuhrt werden. Beispielhaft wird ein Verfahren zur Ruckfuhrung der Biomasse des Gram-negativen Bakteriums Klebsiella planticola in den Fermentationsprozess untersucht und entwickelt.

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the - 10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from - 1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses. (c) 2005 Elsevier B.V All rights reserved.

Die Weise Biotechnologie ist keine neue Disziplin, sondern fasst nur ein Segment der Biotechnologie zusammen, um sie von den beiden anderen grosen Segmenten – der Roten und der Grunen Biotechnologie – abzuheben. Sie ist in den Fokus geraten, weil bahnbrechende Verfahren zur Analyse biologischer Systeme eine Neubetrachtung der Potenziale der Biotechnologie zur Folge haben. Neue Entwicklungen sind abzusehen, wie uber genombasierte Daten mit Hilfe der Bioinformatik, der Molekularbiologie und der Bioverfahrenstechnik Prozesse in ganzheitlicher Betrachtung optimiert werden. Wegen relativ einfacher biologischer Systeme, aber vielfaltiger Anwendungen wird die Weise Biotechnologie als Vorreiterdisziplin ins Feld gefuhrt. Durch die Forderung interdisziplinarer Ansatze zur integralen Prozessentwicklung sollte die biotechnologisch orientierte Industrie in weitem Mase profitieren. Dies gilt insbesondere fur das wissenschaftliche Umfeld und die kleinen und mittleren Unternehmen, die mehr und mehr vom Zugang zu den modernen Methoden der Biotechnologie abhangen. Im internationalen Rahmen ist gerade auf dem Gebiet der Weisen Biotechnologie eine konkurrenzfahige Situation in Deutschland festzustellen. Es bedarf jedoch einer nachhaltigen und langfristigen Unterstutzung, damit sowohl die Wissenschaft als auch die Industrie nicht den Anschluss an die internationale Entwicklung der Weisen Biotechnologie verlieren.

The non-toxic compound N-acetyl-L-phosphinothricin (N-Ac-L-PPT) is used in a so-called deacetylation system to induce male sterility in transgenic plants by tapetum specific deacetylation to the herbicide L-phosphinothricin (L-PPT). A procedure was developed to produce pure racemic and L-isomeric N-Ac-PPT containing less than 30 ppm residual PPT. Experiments applied to wild type tobacco and PPT-resistant tobacco showed that the maximal tolerated N-Ac-PPT concentration would be less than 45 mM of the L-isomer. Otherwise unspecific deacetylation by several acylases, as well as by environmental conditions like higher temperatures or pHs beyond neutrality, increased the residual L-PPT content to toxic concentrations. In contrast, N-acetyl-L-phosphinothricyl-alanyl-alanine (N-Ac-L-PPTT), a substance also occurring during the biosynthesis of phosphinothricyl-alanyl-alanine (PPTT) by some Streptomyces species, was tolerated up to 274 mM by wild type tobacco plants. However, the ArgE deacatylase from Escherichia coli originally used in the deacetylation system, as well as some other acylases, showed no activity towards N-Ac-L-PPTT.

Human Fas ligand (hFasL) is of considerable interest since it is a type II transmembrane glycoprotein that induces programmed cell death, or apoptosis. In this study Dictyostelium discoideum was used to produce a soluble form of the human Fas ligand. The recombinant cells were adapted to a modified synthetic FM medium, called SIH medium. Cells adapted to the SIH medium reached about 2 times higher cell densities and hFasL concentrations on this medium compared with cells growing on the standard complex medium HL-5C. Even higher values were achieved by a dissolved oxygen-controlled fed-batch cultivation in a conventional stirred bioreactor on SIH medium. Cell densities of up to 5.5 x 10(7) ml(-1) and a maximum hFasL concentration of 148 mug l (1) were obtained. These results were further improved by means of continuous cultivation of D. discoideum in a bioreactor equipped with cell retention by microfiltration. At low space velocity very high cell densities of up to 2.4 x 10(8) ml(-1) and hFasL concentrations of up to 205 mug l(-1) were achieved. (C) 2004 Elsevier B.V. All rights reserved.

In this paper the influence of ammonium salt concentration on the production of pharmaceutical grade plasmid DNA from unfed high cell density batch culture on synthetic glycerol media is presented. Ammonium chloride in different concentrations (0 mmol dm(-3), 18.7 mmol dm(-3), 37 mmol dm(-3) and 74 mmol dm(-3) supplemented) was used beside sodium glutamate as nitrogen source. Plasmid DNA concentrations of more than 50 mg dm(-3) were obtained with 37 mmol dm(-3) ammonium. The homogeneity of the DNA produced was confirmed by agarose and capillary gel electrophoresis and found to fulfil the quality requirements set for biopharmaceutical plasmid DNA with more than 90% in the supercoiled form. (C) 2003 Society of Chemical Industry.

Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral vectors like plasmid DNA are currently investigated in various clinical trials. Supercoiled multimeric plasmids are of particular interest for pharmaceutical purpose because they contain multiple copies of a therapeutic gene and can therefore be more efficient vectors. A process for the preparation of Escherichia coli strains replicating dimers, trimers, and tetramers of a 4.6 kb plasmid is presented. Cultivation of these clones on semi-defined glycerol medium in a 7 1 bioreactor shows structural stability of dimers and trimers during the whole cultivation process. Plasmid concentrations and selectivities are compared to the corresponding cultivation with the plasmid monomer. Cultivation of the tetramer replicating strain shows a disintegration of the plasmid multimer and reconstitution of the monomer and smaller multimers. (C) 2003 Elsevier B.V. All rights reserved.

Usually, the fatty acid (FA) composition of lipids from microalgae is determined using samples taken at a single time point only, often without considering the medium composition and cultivation conditions. Therefore, the results only represent the FA composition of cells in a certain growth phase. Furthermore, they may misrepresent the capability of the organism to produce certain FA or lipid mixtures. In this study, 22 FA were analysed quantitatively during the cultivation of Euglena gracilis under different cultivation modes and conditions. For cell growth, various media compositions for heterotrophic and photoheterotrophic conditions were used. Results of extensive FA analysis allowed the determination of appropriate cultivation conditions and durations for yielding lipid mixtures with optimal composition, e.g. for the production of biodiesel or functional food. Drastic differences in the ratio between n3- and n6-polyunsaturated fatty acid (PUFA) ranging from 0.04 to 1.81 were detected. This effect was strongly influenced by the cultivation mode. In addition, media with higher nitrogen concentration resulted in a higher n3/n6-PUFA-ratio as well as improved specific growth rates for all analysed combinations of glucose and nitrogen concentrations. Furthermore, it was demonstrated that the inexpensive proteose peptone medium is ideal for lipid production by E. gracilis. This work provides valuable tools to optimise yield, productivity and n3/n6-PUFA ratio concerning the cultivation of E. gracilis as well as potentially other microalgae in general.

The gene for phytase from Escherichia coli was sequenced and compared with the appA gene. It was found to be a mutant derivative of the appA gene. After fusion with a C-terminal His-tag, phytase was purified by affinity chromatography and the enzymatic properties were analyzed. To develop a system for overexpression and extracellular production of phytase in E. coli, factors affecting the expression and secretion such as promoter type, host strain and selection pressure were analyzed. Using a secretion system based on the controlled expression of the kil gene, the expression of phytase was improved and the enzyme was released into the culture medium at a high level. An effective fermentation strategy based on fed-batch operation was developed.

In this study the use of Euglena gracilis biomass for α-tocopherol, paramylon and biogas production in a value-added chain was investigated. Therefore, we analyzed the dry cell weight and product concentrations at different growth phases during heterotrophic, photoheterotrophic and photoautotrophic cultivation in a low-cost minimal medium. Furthermore, the specific biogas yields for differently derived biomass with and without product recovery were investigated. We demonstrate that growth phase and cultivation mode not only have a significant impact on product formation, but also influence the yield of biogas obtained from anaerobic digestion of Euglena gracilis biomass. The maximum dry cell weight concentration ranged from 12.3±0.14gL(-1) for heterotrophically to 3.4±0.02gL(-1) for photoautotrophically grown Euglena gracilis cells. The heterotrophically grown biomass accumulated product concentrations of 5.3±0.12mgL(-1) of α-tocopherol and 9.3±0.1gL(-1) of paramylon or 805±10.9mL of biogasgvs(-1) (per gram volatile solids). The results for photoautotrophically grown cells were 8.6±0.22mgL(-1) of α-tocopherol and 0.78±0.01gL(-1) of paramylon or 648±7.2mL of biogasgvs(-1). For an energy-saving downstream procedure the extracting agent methanol does not have to be removed strictly. Samples with residual methanol showed a significantly increased biogas yield, because the solvent can be used as an additional substrate for methane production by archaebacteria.

Fixed bed reactors operated by feeding with two immiscible liquids are of increasing importance in chemical as well as biochemical reaction engineering. Based on biocatalysis studies, liquid/liquid/solid three-phase fixed bed reactors have been assessed with respect to the phase distribution in such reactors and the backmixing in each liquid phase. The systems were characterized by the presence of a continuous aqueous phase. The phase distribution in the reactor differed substantially from that in the feed stream, particularly when the feed had a low content of non-polar solvent. The accumulation of the non-polar phase in fixed beds of particulate solids was accompanied by blocking of the interstitial void volume by, and channelling of, the non-polar phase as well as enhanced backmixing of both the aqueous and the non-polar phase. Studies with various nonpolar solvents demonstrated that the choice of organic solvent is important for the operation of three-phase fixed bed reactors. (C) 1998 SCI.

A continuously operated reactor was developed for the limited hydrolysis of whey protein using soluble trypsin. It consisted of a stirred tank followed by a tubular device. A stirred tank was used because it allowes pH control to be achieved more easily. The tubular reactor was applied in order to achieve a high level of conversion without pH control. The tubular reactor was equipped with static mixers for approximating plug-flow behaviour. The behaviour of the reaction system under floating pH in both parts of the reactor was evaluated separately prior to verification of the results in a pilot-plant which consisted of the combination reactor. The operating temperature and pH in the stirred tank were investigated as the main variables of the process. (C) 1998 Elsevier Science Ltd.

Enzymic hydrolyses of naturally occurring protein sources represent complex reactions at a molecular level because of the specificity pattern of proteases and the composition of the substrates involved. In addition, rather global criteria are in practical use for describing the extent of protein hydrolysis. Therefore, mechanistically based kinetic modelling may not be applicable under these circumstances. For practical purposes it is often sufficient to use a descriptive mathematical model, which leads to a reliable interpolation of the experimental data. Empirical models are proposed for the kinetics of the limited tryptic whey-protein hydrolysis. These models are identified by fitting profiles of the fraction of soluble protein (non-protein nitrogen, NPN) as a function of operating time of discontinously operated stirred-tank reactors. Models with at least four parameters are required in order to describe profiles obtained at temperatures at which strong enzyme inactivation is observed. A simple model of only two parameters based on a kinetics with exponential decrease of the activity with increasing fraction of soluble protein is sufficient to simulate the tryptic digestion of whey-protein in batch reactors under conditions of moderate enzyme inactivation applicable for temperatures lower than 60 degrees C. Copyright (C) 1997 Elsevier Science Ltd.

Lipooligosaccharides, synthesized by soil bacteria of the genera Rhizobium, are known to have multifunctional effects on a wide variety of plants as signal substances in symbiosis initiation, cell response elicitation and growth regulation. These so called nodulation (Nod-) factors represent interesting biotechnological products with respect to fundamental studies of symbiotic interactions as well as for potential applications. Therefore, a batch fermentation process on a scale of 30 l has been developed by means of the Rhizobium meliloti strain R.m. 1021 (pEK327) strongly overexpressing the genes for the synthesis of Nod factors. Induction by the flavone luteolin led to growth associated production of the lipooligosaccharides. Ultrafiltration was used for separating the biomass from the filtrate containing the extracellular Nod factors. Simultaneously, ultrafiltration reduced the amount of lipophilic substances, which would otherwise interfere with processes downstream. The second separation step consisted in adsorption on XAD-2, a nonspecific hydrophobic adsorptive resin. Adsorption of Nod factors was carried out by batch operation of a stirred tank. Desorption was performed by elution with methanol in a fixed bed column. A semi-preparative reversed phase HPLC (Polygoprep 100-30 C18) was chosen as the final purification step. The Nod factors were obtained after evaporation and lyophilization. Thus, about 600 mg of Nod factors were produced from 20 l of fermentation broth. The Nod factors produced by Rhizobium meliloti R.m. 1021 (pEK327) were identified by liquid secondary ion mass spectrometry and by reversed-phase HPLC as fluorescent derivatives of 2-aminobenzamide. The biological activity of the products was demonstrated by means of the root hair deformation (HAD-) assay.

The segregational stability of plasmids in a recombinant bioprocess is of extreme significance. Although this is commonly achieved by the selection pressure from antibiotics, their application for the production of therapeutic DNA for gene therapy or for DNA vaccines would be undesirable. Similarly, the presence of antibiotic-resistance genes in the final product would have to be avoided. In addition to the minicircle approach, a type of miniplasmid is able to fulfil the regulatory requirements. The gene tpiA is responsible for the connection of the glycerol metabolic pathway with the essential glycolytic pathway in Escherichia coli. The knockout of genomic tpiA rendered cells completely auxotrophic in minimal medium with glycerol as sole carbon source while allowing growth at a reduced rate with glucose or in complex medium. This was advantageous for optimizing antibiotic-free cloning and selection of recombinant plasmid. Complementation of the auxotrophy by plasmid-borne tpiA led to high segregational and structural plasmid stability, resulting in stable production of a model recombinant enzyme under antibiotic-free conditions in a continuous cultivation. Thus, the complementation of tpiA represents a significant alternative to antibiotics as a selection principle and is therefore of interest for the recombinant production of biotherapeutics in the form of miniplasmids.

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains. However, little is known about the influence of power input and oxygen supply as well as feeding strategies on the production of streptavidin by S. avidinii. This paper provides a systematic analysis of the effect of rotary frequency of the stirrer, leading to a plateau-like streptavidin formation behaviour between 400 and 700 min(-1). This plateau was characterized by specific power inputs between 79 and 107 W L(-1) and corresponding maximal product concentrations of 6.90 μM in 6 days. Lower as well as higher rotary frequencies were not beneficial. Subsequently, a linear fed-batch procedure could be established reproducibly yielding 39.20 μM streptavidin in 14 days, characterized by a constant productivity of 114 nM h(-1). Fed-batch procedures based on dissolved oxygen were less efficient. The linear feeding strategy presented in this paper led to the highest streptavidin concentration ever reported and exceeded the maximal product level given in the literature drastically by a factor of 8.5.

The principle of metal chelate-protein interaction was applied for protein extraction by means of reverse micellar phases. For this purpose, an affinity surfactant was synthesized exposing iminodiacetic acid as the hydrophilic moiety. The application of this substance as a cosurfactant leads to enhanced extraction of proteins exhibiting histidine groups on their surface.

The specific activity of the lipase of Candida rugosa decreases with increasing enzyme concentration even in the presence of soluble substrates. Data about the hydrolysis of 2-chloropropionic acid ethyl ester (CPEE) had suggested that this phenomenon may be caused either by dimerization of the lipase or by adsorption onto the reactor wall. In order to distinguish between both models, experiments were carried out by changing not only the enzyme concentration but also the wetted surface area of the reaction vessel. These novel data reveal that wetted glass surfaces are of only minor importance - if any. Thus, the decrease of activity seems to be caused by some kind of dimerization of the lipase. In addition, it is shown that adsorption onto hydrophobic surfaces can have a dramatic effect on the specific activity. In the presence of large hydrophobic surface areas the specific activity is found to be almost as high as that observed in the presence of insoluble substrate. The analysis of a commonly used test system for lipase activity measurements based on triacetin hydrolysis exhibits a similar activity-enzyme concentration dependence.

Euglena gracilis is shown to be able to grow on potato liquor as the main medium component leading to an interesting biotechnological product represented by paramylon - a beta-1,3-glucan - and, at the same time, revaluating an otherwise annoying waste stream of the potato-starch industry. Paramylon mass fractions of about 75% are obtained for biomass concentrations of 15 g/L during simple batch cultivation under heterotrophic conditions. Supplementation of the growth medium with glucose and the vitamins B-1 and B-12 are shown to improve growth rate as well as paramylon content. E. gracilis grows best at about 27.5 degrees C without requiring pH control.

Since expression profiling methods have been available in a high throughput fashion, the implication of these technologies in the field of biotechnology has increased dramatically. Microarray technology is one such unique and efficient methodology for simultaneous exploration of expression levels of numerous genes. Likewise, two-dimensional gel electrophoresis or multidimensional liquid chromatography coupled with mass spectrometry are extensively utilised for studying expression levels of numerous proteins. In the field of biotechnology these highly parallel analytical methods have paved the way to study and understand various biological phenomena depending on expression patterns. The next phenomenological level is represented by the metabolome and the (metabolic) fluxome. However, this chapter reviews gene and protein profiling and their impact on understanding recombinant protein production. We focus on the computational methods utilised for the analyses of data obtained from these profiling technologies as well as prominent results focusing on recombinant protein expression with Escherichia coli. Owing to the knowledge accumulated with respect to cellular signals triggered during recombinant protein production, this field is on the way to design strategies for developing improved processes. Both gene and protein profiling have exhibited a handful of functional categories to concentrate on in order to identify target genes and proteins, respectively, involved in the signalling network with major impact on recombinant protein production.

Online monitoring of cell density and cell viability is a challenging but essential task to control and optimize biotechnical processes and is of particular interest for the growing field of animal cell cultures. For this purpose, we introduce an optical approach for automated cell detection and viability classification of suspended mammalian cells. Our proposed system CellViCAM is capable of evaluating dark field micrographs by means of several image processing and supervised machine learning techniques without the use of any dyes or fluorescent labeling. Using a human cell line as the reference culture, an efficient cell detection procedure has been established also enabling a cell density estimation. Furthermore, a comprehensive but reagent-free viability analysis, based on a semi-automatic training data generation, has been developed. By means of an extensive validation dataset we can show that the CellViCAM approach can be considered as an equivalent to staining-based methods and moreover, how it provides a technical platform for a more differentiated cell state classification into living, necrotic, early and late apoptosis. (C) 2010 Elsevier B.V. All rights reserved.

Aiming to facilitate the accessibility of recombinant proteins produced with Escherichia coli, extracellular expression may be achieved by means of bacteriocin release protein (BRP) coexpression. Different types of BRPs were tested in order to optimize protein secretion into the culture medium. Those included the well-studied BRPs of the Colicin E1 and Cloacin DF13 bacteriocins and variants thereof. BRP expression was stringently controlled by means of the arabinose inducible P-BAD promoter, which accounts for a broad-range adjustment of expression strength. Using appropriate arabinose concentrations, a concentration range was determined, that allowed efficient secretion of the model proteins alkaline phosphatase and beta-lactamase, with 90-95% of the proteins released into the culture medium. Kinetic investigations into BRP expression and protein secretion revealed a rapid increase of extracellular protein concentration within 5-10 min past induction. Alternatively to fine-tuned BRP expression during cultivation, protein production and secretion could be decoupled by establishment of appropriate induction strategies and up to 90% of alkaline phosphatase was released into the culture medium within 3 h after reaching maximum biomasss concentrations. Both, fine-tuned and growth decoupled BRP expression accounted for extracellular alkaline phosphatase concentrations of roughly 500 mgl(-1) of culture broth and selectivities of 50 mg of this enzyme per gram of cell dry mass, respectively. (C) 2009 Elsevier B.V. All rights reserved.

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (> 200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g(-1) of plasmid DNA with almost complete plasmid recovery.

Euglena gracilis was cultivated on a synthetic medium of well-defined components. Biomass and paramylon (beta-1, 3-glucan) concentrations were the most important variables monitored. Mass production in the bioreactor was carried out following studies of operating conditions in shaken flasks. E. gracilis grew best at about 30 ^oC and at a low pH of 3. A pH control was not necessary, although the pH increased considerably at the end of the cultivation processes. Aeration could be performed at low stirrer frequency. Biomass concentrations of about 13– 14 g/L were obtained with paramylon mass fractions of 50– 60%, by starting with a synthetic medium containing 15 g/L of glucose as the main carbon source.

Functionalized linear dextrins were sepd. on a preparative scale by HPLC on RP-18 phases with water as the eluent. These dextrins were obtained by the transfer of a-cyclodextrin to glucosides by means of a cyclodextrin-glycosyltransferase of Klebsiella pneumoniae M5 aI. Product recovery was readily achieved owing to the simple eluent used. [on SciFinder (R)]

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.

Transcript and metabolite abundance changes were analyzed in evolved and ancestor strains of Escherichia coli in three different evolutionary conditions, Background Evolutionary changes that are due to different environmental conditions can be examined based on the various molecular aspects that constitute a cell, namely transcript, protein, or metabolite abundance. We analyzed changes in transcript and metabolite abundance in evolved and ancestor strains in three different evolutionary conditions - excess nutrient adaptation, prolonged stationary phase adaptation, and adaptation because of environmental shift - in two different strains of bacterium Escherichia coli K-12 (MG1655 and DH10B). Results Metabolite profiling of 84 identified metabolites revealed that most of the metabolites involved in the tricarboxylic acid cycle and nucleotide metabolism were altered in both of the excess nutrient evolved lines. Gene expression profiling using whole genome microarray with 4,288 open reading frames revealed over-representation of the transport functional category in all evolved lines. Excess nutrient adapted lines were found to exhibit greater degrees of positive correlation, indicating parallelism between ancestor and evolved lines, when compared with prolonged stationary phase adapted lines. Gene-metabolite correlation network analysis revealed over-representation of membrane-associated functional categories. Proteome analysis revealed the major role played by outer membrane proteins in adaptive evolution. GltB, LamB and YaeT proteins in excess nutrient lines, and FepA, CirA, OmpC and OmpA in prolonged stationary phase lines were found to be differentially over-expressed. Conclusion In summary, we report the vital involvement of energy metabolism and membrane-associated functional categories in all of the evolutionary conditions examined in this study within the context of transcript, outer membrane protein, and metabolite levels. These initial data obtained may help to enhance our understanding of the evolutionary process from a systems biology perspective.

Exptl. investigations of the thermal bulk polymn. of styrene (I) in pilot plants of different sizes were done. Each pilot plant consisted of a tubular recycle reactor connected in series with a tubular reactor, both completely filled with Sulzer motionless mixers. Kinetic, reactor, and viscosity models were verified at I conversions ?96%, temps. ?210 Deg, and polystyrene mol. wts. ?360,000. Scale-up studies were done which confirmed that multitubular reactors of special design could be applied for industrial polymn. processes. [on SciFinder (R)]

Fermentation industries require in-situ real-time monitoring of cell viability during fermentation processes. For this purpose, reagent-free approaches are desired because they can be used for in situ analysis and reduce the system's complexity. We have developed an automatic way of determining cell viability via analysis of time-lapse image sequences taken by dark field microscopy without the aid of any additional reagents. The image processing is based on neural networks based machine vision, involving Principal Component Analysis (PCA) to investigate the dynamic information of intracellular movements. In consequence, the essential features as the vital sign of the target cells are discovered. Viability predictions using the Support Vector Machine (SVM) classifier have been done successfully on the datasets with different qualities. Accuracy up to above 90% has been obtained on the basis of image enhancement. Robustness of the system is proved by the results of the tests. The model organism we have used is Saccharomyces cerevisiae, however, this technique can promisingly be applied for the identification of cell viability of other organisms as well.