Biomedical subjects

C Wandrey

Publications and source records attributed to C Wandrey.

At least 19 recordsLinked to original sources

PubMed2005

Synthesis of enantiopure (5R)-hydroxyhexane-2-one with immobilised whole cells of Lactobacillus kefiri.

Whole-cell reduction of (2,5)-hexanedione to yield highly enantiopure (5R)-hydroxyhexane-2-one (enantiomeric excess >99%) with Lactobacillus kefiri DSM 20587 was investigated. Cell immobilisation with sodium cellulose sulphate was chosen as the most suitable encapsulation matrix, giving an immobilisation yield of 40%. Despite the lowered biocatalytic activity from cell immobilisation, the bioreduction process was vastly improved with the help of reaction engineering techniques (batch to a plug flow reactor set-up). High selectivity (95%) and space-time yield (87 g L(-1) day(-1)) were achieved in the plug flow reactor. The biocatalyst remained active (68% residual activity) after 6 days of operation.

Bioreactors↗

PubMed2004

Modeling of the pyruvate production with Escherichia coli in a fed-batch bioreactor.

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q(VG)=10 mL h(-1). Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q(VG)=20 and 30 mL h(-1), respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking-Piret/Levenspiel term).

Bioreactors↗

PubMed2004

Indirect electrochemical reduction of nicotinamide coenzymes.

Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2'-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1-10 mM NAD(P)(+) and 2.5-200 microM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)(+) was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.

Adsorption↗

PubMed2004

Short-term immunosuppression reduces fibrotic cellular infiltration around barium-M-alginate microbeads injected intraportally.

INTRODUCTION: One of the major barriers affecting the viability of encapsulated islets is pericapsular fibrotic infiltration (PFI). This study aimed to design strategies to reduce PFI around intraportally injected alginate microbeads. METHODS: Empty, highly purified, barium-M-alginate microbeads (400 microm) were injected intraportally into Lewis rats (3000 beads/rat). Rats (n = 9/group) were treated daily with either rapamycine (RAPA; 1 mg/kg/d p.o.), tacrolimus (TAC; 2 mg/kg/d p.o.), a combination of both, or gadolinium-chloride (GdC13, 20 mg/kg/d i.v., at day -1 and day +4). Treatment was discontinued at 10 days. Three rats/group were sacrificed at 3, 7, and 42 days after transplantation. Cellular composition of PFI was evaluated by immunohistochemistry. Severity of the reaction to the beads was determined by measuring the thickness of PFI on histology. RESULTS: The main cellular components of PFI in the liver were macrophages and myofibroblasts. There was a significant (P <.05) reduction in the thickness of PFI in all treated groups, even 6 weeks after transplantation. Encapsulated rat islets showed excellent insulin response to glucose in vitro, with a stimulation index of 3.6 +/- 2.0. CONCLUSION: Combination of highly purified alginate with short-term immunosuppression reduces fibrotic overgrowth around microbeads injected intraportally.

Alginates↗

PubMed2003

Continuous gluconic acid production by isolated yeast-like mould strains of Aureobasidium pullulans.

By extensive microbial screening, about 50 strains with the ability to secrete gluconic acid were isolated from wild flowers. The strains belong to the yeast-like mould Aureobasidium pullulans (de Bary) Arnaud. In shake flask experiments, gluconic acid concentrations between 23 and 140 g/l were produced within 2 days using a mineral medium. In batch experiments, various important fermentation parameters influencing gluconic acid production by A. pullulans isolate 70 (DSM 7085) were identified. Continuous production of gluconic acid with free-growing cells of the isolated yeast-like microorganisms was studied. About 260 g/l gluconic acid at total glucose conversion could be achieved using continuous stirred tank reactors in defined media with residence times (RT) of about 26 h. The highest space-time-yield of 19.3 g l(-1) x h(-1)) with a gluconic acid concentration of 207.5 g/l was achieved with a RT of 10.8 h. The possibility of gluconic acid production with biomass retention by immobilised cells on porous sinter glass is discussed. The new continuous gluconate fermentation process provides significant advantages over traditional discontinuous operation employing Aspergillus niger. The aim of this work was the development of a continuous fermentation process for the production of gluconic acid. Process control becomes easier, offering constant product quality and quantity.

Ascomycota↗

PubMed2002

Citric acid production by Candida strains under intracellular nitrogen limitation.

A suitable strain and important factors influencing citric acid formation in yeasts were identified. Candida oleophila ATCC 20177 was chosen as the best citric acid producer from several Candida strains. Yields of 50 g/l citric acid were produced in shake flask and 80 g/l in fed-batch fermentations with 1.5 and 3 g/l NH(4)Cl under non-optimized conditions. Ammonium nitrogen was identified as the limiting substrate for citrate formation. Citric acid excretion begins a few hours after exhaustion of nitrogen in the medium. The importance of intracellular nitrogen limitation was clarified by elemental analysis of C. oleophila biomass. The nitrogen content of C. oleophila biomass decreased from 7.45% during the growth phase to 3.96% in the production phase. The biomass contained less carbon and more trace elements in the growth phase compared with the production phase. Relatively high intracellular NH(4)(+) concentration of about 1.2 mg/g biomass (~37.4 mM) was found during the production phase. The low intracellular nitrogen content and increase of intracellular NH(4)(+) concentration, possibly caused by proteolysis following extracellular nitrogen exhaustion, trigger citric acid production. Intracellular nitrogen limitation and the increase in intracellular NH(4)(+) concentration are the most important factors influencing citric acid formation in yeasts.

Biomass↗

PubMed2001

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.

Biochemistry↗

PubMed2001

Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.

The quantitative comprehension of microbial metabolic networks is a prerequisite for an efficient rational strain improvement ("metabolic engineering"). It is therefore necessary to accurately determine the concentration of a large number of reactants (i.e., metabolites, nucleotides, cofactors) in order to understand "in vivo" reaction kinetics. Quantification of intracellular concentrations of glycolytic intermediates and nucleotides in Escherichia coli K12 using a perchloric acid extraction and an LC-ESI-MS method was achieved. Intracellular metabolites (e.g., glucose 6-phosphate, fructose 1,6-bisphosphate, 6-phospho gluconate, acetyl-CoA, adenine nucleotides) were quantified under defined (glucose-limited steady-state) growth conditions. The method was verified by comparing the intracellular metabolite concentrations measured via LC-ESI-MS with enzymatic determinations. It is thus possible to identify and quantify more than 15 intracellular metabolites in parallel with a minimal amount of sample volume.

Acetyl Coenzyme A↗

PubMed2001

Evaluation of parallel operated small-scale bubble columns for microbial process development using Staphylococcus carnosus.

Shake flasks and pH-controlled small-scale bubble columns were compared with respect to their usefulness as a basic tool for process development for human calcitonin precursor fusion-protein production with Staphylococcus carnosus. Parallel control of the pH (and making use of the base addition data) is necessary to study the effects of medium composition, to identify pH-optima and to develop a medium, which minimizes the acid excretion of S. carnosus. This medium with glycerol as energy source and yeast extract as carbon and nitrogen source resulted in cell dry weight concentration in shake flasks of 5 g l(-1), which were thus improved by a factor of 10. Cell dry weight concentrations of up to 12.5 g l(-1) were measured in the batch process with pH-controlled small-scale bubble columns due to their higher oxygen transfer capability. In contrast to shake flasks it was demonstrated, that the batch process performance of recombinant S. carnosus secreting the human calcitonin precursor fusion-protein was identical within the estimation error in pH-controlled small-scale bubble columns compared to the stirred-tank reactor.

Cell Division↗

PubMed2001

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor.

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.

Amines↗

PubMed2001

Purification of polymeric biomaterials.

Employing a combined filtration and precipitation method, the endotoxin concentration in sodium alginate (SA) and sodium cellulose sulfate (SCS) was reduced to a value of 200 EU/g polymer. This is one tenth of the regulatory threshold calculated, for example, for an appropriate bioartificial pancreas that consists of approximately 420,000 encapsulated islets of Langerhans. The low endotoxin (ET) levels were maintained below this threshold during a six-month storage period. The purification procedure of the polymers did not negatively influence the final microcapsule properties. The mechanical stability of microcapsules from purified material is even slightly higher than that of microcapsules from the original polymers. A second approach to avoid endotoxin release from the device is its direct complexation during the bead or capsule formation process. The durability of endotoxin binding in binary, ternary, and quaternary complexes could be demonstrated for storage in culture medium and saline. Very low total endotoxin release from the complexes was detected after three months in culture medium and five months in saline. This complexation is primarily based on electrostatic interactions with the participating cationic components and provides additional security for the final bioartificial organ or delivery device.

Alginates↗

PubMed2000

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Reprinted from Biotechnology and Bioengineering, Vol. XXIII, No. 12, Pages 2789-2802 (1981).

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10(4) [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L-leucine dehydrogenase (LEUDH) was used as catalyst for the reductive amination of alpha-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L-leucine. Formate dehydrogenase (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L-leucine. The kinetic constants V(max) and k(m) of the enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L-leucine was produced continuously out of alpha-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).

Bioreactors↗

PubMed2000

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

Amino Acids↗

PubMed1999

Polycations as displacer in high-performance bioseparation.

Displacement chromatography is an interesting but up to now rarely used type of preparative biochromatography. The lack of well-engineered and accessible displacer contributes to this phenomenon. In this paper a novel type of displacer is introduced for cation-exchange displacement chromatography, which will soon become commercially available. The molecule is a well-defined PolyDADMAC [poly(diallyldimethylammonium chloride)] with a molar mass of less than 35000 g/mol, an exclusively linear structure and a molar mass polydispersity of less than 1.5. A method for synthesizing such a polymer at high yields is described. The PolyDADMAC is shown to be an efficient displacer of basic proteins from strong cation-exchange columns.

Chromatography, Ion Exchange↗

PubMed1999

New multicomponent capsules for immunoisolation.

A new generation of microcapsules based on the use of oligomers which participate in polyelectrolyte complexation reactions has been developed. These freeze-thaw stable capsules have been applied as a bioartificial pancreas and have resulted in normoglycemia for periods of six months in concordant xenotransplantations. The new chemistry permits the control of permeability and mechanical properties over a wide range and can be adapted both to microcapsule and hollow fiber geometries rendering it a robust tool for encapsulation in general. Methods, and metrics, for the characterization of the mechanical properties and permeability of microcapsules are presented.

Artificial Organs↗

PubMed1998

In situ generated O-glycan core 1 structure as substrate for Gal(beta 1-3)GalNAc beta-1,6-GlcNAc transferase.

beta-Galactosidase from bovine testes was used in a one pot reaction together with a recombinant beta-1,6-GlcNAc transferase for the synthesis of GlcNAc(beta 1-6)GalNAc(alpha 1-OBn) (core 6-Bn). The galactosidase, which reversibly links galactose via a (beta 1-3) linkage to N-acetylgalactosamine, provides the substrate for the GlcNAc transferase in situ. The synthesis was carried out with a yield > 90%.

Animals↗

PubMed1997

Purification and characterization of an alcohol:N,N-dimethyl-4-nitrosoaniline oxidoreductase from the methanogen Methanosarcina barkeri DSM 804 strain Fusaro.

Cell-free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to approximate homogeneity by column chromatography. It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa. The purified protein had an isoelectric point of 4.3. It possesses a tightly but noncovalently bound NADP(H) cofactor. Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium. The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol). NDMA-ADH from M. barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier. The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis. An alignment of the N-terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity. Nevertheless there was a quite interesting sequence similarity in the first 30 N-terminal amino acids to plant cinnamyl alcohol dehydrogenase. NDMA-ADH from M. barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.

Alcohol Oxidoreductases↗

PubMed1997

Pilot scale expression and purification of soluble protein A tagged beta 1,6N-acetylglucosaminyltransferase in CHO cells.

Expression of recombinant soluble protein A tagged mouse core 2 beta 6-N-acetylglucosaminyltransferase (E.C. 2.4.1.102) has been scaled-up in CHO cells using a continuously operating fluidized bed system yielding 0.3 U/day. A one step 213 fold purification by affinity chromatography on IgG-Sepharose yielded a stable enzyme preparation with a specific activity of 44 mU/mg. The enzyme was shown to belong to the L-type with a highly restricted specificity for the acceptor substrate Gal beta 1-->3GalNAc alpha 1-->R (core 1). Only little activity towards GlcNAc beta 1-->3Gal-NAc alpha 1-->R (core 3) (< 1%) and no incorporation on unsubstituted benzyl or peptide-bound GalNAc was detected. Zn2+ and to a lesser extent Mn2+ were found to be inhibitory whereas Mg2+ could activate the enzyme. The enzyme preparation proved suitable for in vitro application as a catalyst for the synthesis of core 2 structures.

Animals↗