Biomedical subjects

W D Deckwer

Publications and source records attributed to W D Deckwer.

At least 37 records · Page 2Linked to original sources

PubMed1997

Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae.

Stoichiometric analysis of pathways involved in anaerobic bioconversion of glycerol by Klebsiella pneumoniae revealed that enzyme(s) in addition to pyruvate formate-lyase (PFL) must be involved in pyruvate decarboxylation. In this work, enzymatic evidence is presented that confirmed a simultaneous involvement of pyruvate dehydrogenase complex (PDH) and excluded the presence of pyruvate:ferredoxin oxidoreductase in this anaerobic bioprocess. The in vitro PDH activity of cell extract from continuous culture was found to be strongly affected by the substrate (glycerol) concentration in medium and cell growth rate (dilution rate). It increases with increasing glycerol concentration and correlates well with the specific substrate uptake rate at different dilution rates in a kind of saturation function. At a similar substrate uptake rate, it decreases with cell growth rate. The in vitro activity of PDH is much higher than its in vivo activity calculated from the pathway stoichiometry but comparable to the calculated in vivo activity of PFL.

Anaerobiosis↗

PubMed1997

Membrane adsorbers for selective removal of bacterial endotoxin.

Surface-modified flat-sheet microfiltration membranes were functionalised with poly-L-lysine, polymyxin B, poly(ethyleneimine), L-histidine, histamine, alpha-amylase and DEAE as well as deoxycholate. Their suitability to remove endotoxin from both buffers and protein solutions was examined using bovine serum albumin, murine IgG1 and lysozyme as model proteins. In protein-free solutions reduction from 6000 EU/ml to <0.1 EU/ml was achieved with all applied ligands; only alpha-amylase as well as L-histidine and histamine, when immobilized via the non-ionic spacer bisoxirane, exhibited low clearance factors at neutral pH. The adsorption of endotoxin is mainly ruled by electrostatic interaction forces. Thus in multi-component systems, such as endotoxin-contaminated protein solutions, competing interactions take place: acidic proteins compete with endotoxin for binding sites at the membrane adsorbers, basic proteins compete with the ligands for endotoxin and act as endotoxin carriers. With properly chosen conditions the membrane adsorbers presented here show exceptional effectiveness also in the presence of proteins. They are generally superior to functionalised Sepharose chromatographic sorbents and allow fast processing. They may contribute to reduce the risks in the application of parenterals and diagnostics.

Adsorption↗

PubMed1996

Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations.

Clavulanic acid (CA) was produced by Streptomyces clavuligerus in medium containing glycerol and soy meal or soy meal extract. With regard to growth and CA productivity, the microorganism showed significant differences if solid soy meal as such or its extract were applied as the major nitrogen source. If the extract is used, growth and CA production take place simultaneously and in the stationary phase the CA concentration is stagnant or reduces. If soy meal is used, growth is threefold faster and CA is only generated in the stationary phase. In the case of using the soy meal extract, the decrease of the CA concentration is mainly due to decomposition or re-metabolisation of CA in the presence of the microorganism. This conclusion is supported by in vivo and in vitro data on CA decomposition.

Anti-Bacterial Agents↗

PubMed1996

Purification of murine IgG1 on group specific affinity sorbents.

Murine IgG1 a CD4-antibody, was produced from the cell line MAX 16 H 5. DEAE-Sepharose CL-6B and group specific sorbents based on different ligands, such as protein A-Sepharose, AvidChrom, Thiophilic Resin (T-Resin) Fractogel EMD TA 650S, Sepharose 4B-immobilized metal chelate and histidine sorbents, were compared in terms of their suitability for the large-scale purification of this IgG. Evaluation criteria were selectivity for IgG1 protein capacity and recovery, stability under cleaning-in-place (CIP) conditions and the rate of IgG1 adsorption. Both thiophilic gels performed best under these conditions, with Fractogel EMD TA 650S demonstrating slightly higher contamination with other proteins compared to the T-Resin based on 6% beaded Agarose. Higher purities were obtained with these gels compared to conventional purification techniques employing a combination of ion exchange and hydrophobic interaction chromatography. Favorable adsorption kinetics were found on DEAE-Sepharose with the effective diffusivity in the pores of the sorbent being five-times accelerated compared to the diffusion coefficient of IgG1 in solution. Multilayer adsorption of the IgG1 was experienced from adsorption isotherms on DEAE-Sepharose and Fractogel EMD TA 650S. This was linked to an early breakthrough of this protein during frontal chromatography. Co-purification of pyrogens between two-(Thiophilic Resin) and five-times (DEAE-Sepharose) of the original level was significant with all sorbents except protein A-Sepharose and AvidChrom. Thus an additional chromatographic step is required in case of pyrogen contamination of the cell culture homogenate.

Animals↗

PubMed1995

Comparison of the energetic efficiencies of hydrogen and oxychemicals formation in Klebsiella pneumoniae and Clostridium butyricum during anaerobic growth on glycerol.

Data for the anaerobic growth of Klebsiella pneumoniae DSM 2026 and Clostridium butyricum DSM 5431 on glycerol have been analyzed using the concept of material and available electron balances with consideration for hydrogen production. Models for the kinetics of energetic efficiencies of product formation under low residual glycerol are presented. For Klebsiella pneumoniae, the specific rates of electron transfer to the products were mainly significantly dependent on specific growth rate with the exception of ethanol and hydrogen which were also significantly non-growth associated. In the case of Clostridium butyricum, the rates were only growth rate dependent, except for hydrogen formation. The analysis also indicated that the production of 1,3-propanediol by Klebsiella pneumoniae was favoured by limitations other than glycerol limitation, while hydrogen generation was best under low residual glycerol and particularly in the presence of external 1,3-propanediol. Klebsiella pneumoniae appeared to be able to incorporate more of the available electrons of glycerol into hydrogen as compared with the Clostridium butyricum. The study demonstrates the need for properly considering H2 in models describing anaerobic processes.

Clostridium↗

PubMed1995

Simple fed-batch technique for high cell density cultivation of Escherichia coli.

A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed. A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (mu < mu crit). Cell concentrations of 128 and 148 g per 1 dry cell weight (g l-1 DCW) were obtained using glucose or glycerol as carbon source, respectively.

Bacteriological Techniques↗

PubMed1995

Growth inhibition by ammonia and use of a pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum.

The inhibitory effect of ammonia on the growth of the polychlorinated xenobiotic-degrading bacterium Mycobacterium chlorophenolicum was examined. The strain is inhibited by both the ionized and nonionized forms of ammonia, At pH 6.9, 50% reduction of the growth rate was observed at 6.8 g/l total ammonium. For 23 experiments performed in shake-flask culture at different pH values, and ammonium concentrations a growth model based on the extended Monod kinetic fits the data with a deviation of 5.3%. To overcome growth inhibition in bioreactors, a pH- controlled feeding strategy was developed for effective cultivation of M. chlorophenolicum at an ammonium level below 0.3 g/l. The ammonium addition was controlled online by the stoichiometric interdependence of ammonium consumption and pH decline. With this online control strategy, a biomass concentration as high as 26.2 g/l can be achieved within less than a week of cultivation. The yield is also increased from 0.32 g to 0.43 g biomass (per gram glucose). The strategy developed provides an effective method for the production of biomass o M. chlorophenolicum serving as the inoculum in remediation technologies.

Automation↗

PubMed1994

Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1.

Continuous and fed-batch cultures of recombinant Escherichia coli TG1 were carried out in order to study plasmid stability and recombinant product formation at different specific growth rates. The aprotinin::beta-galactosidase gene (Ap::lacZ) was placed under the control of two different promoter/repressor systems, the PLac/lacI (pPLac8) and the lambda PL/cIts857 (pPL6) system. The chemically (0.5 mM IPTG) induced gene expression exhibited higher product activity and plasmid stability than the thermally (40 degrees C) induced expression. In fed-batch cultivations with the more stable E. coli TG1(pPLac8) a special feeding strategy allowed bacterial growth with a constant growth rate mu for several hours up to high cell densities. The cloned gene product activity was noticeably effected by the specific growth rate and the cell density at the moment of induction. In particular, the enzyme activities passed a pronounced maximum value in dependence of the set growth rate. The results indicate that fed-batch cultivation strategies are well suited to produce recombinant gene products.

Escherichia coli↗

PubMed1994

Production of human parathyroid hormone by recombinant Escherichia coli TG1 on synthetic medium.

Production of human parathyroid hormone (hPTH) by Escherichia coli TG1:I52cIts was studied. The hPTH is expressed as a fusion protein under control of the bacteriophage lambda pR promoter. The organism grows on glucose/mineral salt medium and the expression of the gene product was investigated under variation of temperature and growth rate prior to and after induction. hPTH formation largely depends on cultivation temperature and is optimal for a temperature shift from 30 to 38 degrees C. Product expression is growth coupled and specific hPTH concentration is independent of growth rate. The results are compared with a previous study on E. coli N4830:pEX-PPTH grown on complex media.

Culture Media↗

PubMed1994

Structure and physical properties of the extracellular polysaccharide PS-P4 produced by Sphingomonas paucimobilis P4 (DSM 6418).

A new strain, Sphingomonas paucimobilis P4 (DSM 6418), was found during a screening programme for exopolysaccharide-producing bacteria. The highly viscous fermentation broth yields a polysaccharide (up to 10 kg/m3), named PS-P4, and shows thixotropic flow behaviour. In the presence of phosphate ions, PS-P4 forms aqueous gels after heating and cooling at alkaline pH. After isolation and purification of the exopolysaccharide, structural analysis by 1D and 2D 1H NMR spectroscopy and mass spectrometry was performed. The deacylated exopolysaccharide has the following repeating trisaccharide structure:-->4)-beta-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D- Glcp(1-->Additionally, the presence of ester-bound acetic acid, D-glyceric acid, and (R)-3-hydroxybutyric acid in the native polysaccharide was demonstrated.

Carbohydrate Sequence↗

PubMed1994

Pseudomonas putida Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants.

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudomonas putida with a minitransposon containing merTPAB, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg, at greater rates. Constitutive overexpression of merTPAB had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.

Journal Article↗

PubMed1993

Studies on the production of human parathyroid hormone by recombinant Escherichia coli.

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830:pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage lambda PR promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30 degrees C; the temperature shift to induce production of fusion protein was varied from 36 to 42 degrees C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38 degrees C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/1 per hour). By applying a fed-batch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.

Biotechnology↗

PubMed1993

Kinetic analysis of cellular nucleotides correlated to growth and tPA production in perfusion cultures of human fibroblast cells.

A maximum cell density of 8 x 10(6) viable human fibroblast cells/ml was obtained on microcarriers at a perfusion rate of 0.6 mL/min-a rate which maintained a quasi-steady state. The maximum tPA production was approximately 1.2 micrograms/ml and obtained when the cell density was relatively constant during the later periods of cultivation. Specific UTP and tPA production rates had a correlation factor of 0.85, and cell growth to oxygen consumption had an 0.92 correlation factor. ATP generation was strongly correlated to glutamine consumption, with a lower correlation to oxygen utilization.

Adenosine Triphosphate↗

PubMed1993

Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria.

The microorganisms used for the mercury retention experiments were natural isolates and genetically engineered bacteria. All mercury-resistant strains contained the merA gene. Column experiments with these strains were carried out by immobilizing them on different support materials. To obtain kinetic data of the reductase activity for whole cells and the crude extract, batch experiments were carried out under different conditions.

Aeromonas↗

PubMed1992

Enzyme purification by immobilized metal ion affinity partitioning--application to D-hydroxyisocaproate dehydrogenase.

Extraction and purification of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei has been studied by means of immobilized metal ion affinity partitioning (IMAP) in aqueous two-phase systems. The partition of the enzyme can be influenced strongly by inclusion of iminodiacetic acid as chelating ligand coupled to polyethylene glycol and loaded with Cu2+ ions into the phase system. This applies to polyethylene glycol/dextran as well as polyethylene glycol/salt phase systems. An increase in enzyme partition coefficient of up to about 1000-fold was observed. Based on the mathematic model presented recently by Suh and Arnold (1990) approximately 6.4 histidine residues were calculated to be involved in the enzyme-metal chelate complex. Direct extraction of the enzyme from both cell homogenate and cell debris supernatant proved unsatisfactory due to disturbances caused by the presence of cell debris and low molecular weight cell components. A combination with a preceding prepurification by a fractional precipitation with polyethylene glycol resulted in a strong affinity effect accompanied by an efficient purification during IMAP (purification factor of 11 with a yield of approximately 90%). Based on this step, an efficient downstream process can be designed for D-hydroxyisocaproate dehydrogenase.

Alcohol Oxidoreductases↗

PubMed1991

High cell density fermentation of recombinant Escherichia coli with computer-controlled optimal growth rate.

In recent years recombinant DNA technology has enabled us to produce various proteins of therapeutic importance with microorganisms. As an appropriate host organism, E. coli plays a dominant role. Yields of E. coli dry cell mass in shaker flask culture range from 1-2 g/L, whereas in fermentors up to 10 g dry cells/L can be achieved. ZIMET and GBF have developed a high cell density fermentation process that produces E. coli (on a glucose/mineral salt medium) up to more than 100 g dry cells/L in a special fed-batch mode. This cultivation strategy prevents oxygen limitation and hence the accumulation of acetate and other metabolic byproducts. The specific growth rate can be adjusted so that product formation reaches its optimum value. An example of the production of alpha1-interferon is presented. The high cell density fermentations were realized in 30- and 450-L Chemap fermentors (ZIMET) and in a three-stage bioreactor scale-up system (72, 300, and 1,500 L) developed in cooperation with GBF and B. Braun Melsungen AG. Multiloop controllers were used to control the process variables.

Biotechnology↗

PubMed1991

Studies on the production of lipase from recombinant Staphylococcus carnosus.

Production of lipase from recombinant Staphylococcus carnosus pLipPS1 was studied in standard stirred tank bioreactors. Only low lipase activity was obtained under conventional operating conditions, i.e., moderate to high stirring speeds and aeration rates for keeping the dissolved oxygen concentration at high levels. Additional targetted experiments indicated that the reason for the observed low lipase activity is lipase inactivation due to surface forces and shear stress at the gas/liquid interface. Therefore, a cultivation strategy is proposed that minimizes gas/liquid interfacial area and maximizes the driving concentration for O2 mass transfer by controlling the dissolved oxygen to low values by gentle stirring and low aeration rates. Thus, high lipase activities can be obtained even in larger scale standard stirred tank bioreactors.

Fermentation↗

PubMed1991

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.

Bacteriological Techniques↗