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Biomedical subjects

E J Vandamme

Publications and source records attributed to E J Vandamme.

At least 19 recordsLinked to original sources
PubMed2006

Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures.

E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase (gltA) or phosphoenolpyruvate carboxylase (ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 and 23 g/l, respectively).

Acetates↗
PubMed2006

Transport kinetics of ectoine, an osmolyte produced by Brevibacterium epidermis.

Brevibacterium epidermis DSM 20659 is a halotolerant Gram-positive bacterium which can synthesize the osmolyte, ectoine, but prefers to take it up from its environment. The present study revealed that B. epidermis is equipped with at least one transport system for ectoine, with a maximal transport velocity of 15.7 +/- 4.3 nmol/g CDW.min. The transport requires energy (ATP) and is completely inhibited by the proton uncoupler, CCCP. The ectoine uptake system is constitutively expressed at a basal level of activity and its activity is immediately 10-fold increased by hyper-osmotic stress. Initial uptake rates are not influenced by the intensity of the hyper-osmotic shock but the duration of the increased activity of the uptake system could be directly related to the osmotic strength of the assay solution. Competition assays indicate that betaine, but not proline, is also transported by the ectoine uptake system.

Amino Acids, Diamino↗
PubMed2003

Dynamics and optimal conditions of intracellular ectoine accumulation in Brevibacterium sp.

The optimal conditions for the intracellular synthesis of ectoine were determined in a halotolerant Brevibacterium sp. The size of the intracellular ectoine pool in the bacterial cells is shown to depend on the external salt concentrations, type of carbon source and aeration level. In erlenmeyer flasks a maximum concentration of intracellular ectoine of about 0.9 g/l was obtained. Under controlled aeration in a 1.5 l fermentor this level could be increased to 1.2 g/l. Consecutive cell transfers to media with increasingly higher salt concentrations enabled us to reach even higher levels, up to 1.6 g/l on erlenmeyer scale. The ectoine synthesis takes place immediately after the osmotic upshock. Within one generation time, the new corresponding specific intracellular ectoine concentration is reached.

Amino Acids, Diamino↗
PubMed2003

Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration.

D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases. For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used. Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase. When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol. To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability. As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C. Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed. Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

Culture Media↗
PubMed2003

Enzymatic conversion of the clavan exopolysaccharide by Streptomyces sp. YSDL-20.

A screening programme was set up to isolate microorganisms able to hydrolyse the complex biopolymer clavan produced by Clavibacter michiganensis subsp. michiganensis LMG 5604. This valuable exopolysaccharide is very rich in L-fucose (37.5% w/w), a rare sugar, used in the medical field (Vanhooren, 1999). A microorganism capable of depolymerizing the polymer may decrease the high viscosity during clavan batch fermentations and remove the limitations of the oxygen transfer and consequently increase the clavan yield. It could also release free L-fucose or L-fucose rich oligosaccharides. An actinomycete, designated YSDL-20, isolated from a soil sample, was able to depolymerize this biopolymer. Based on its morphology and molecular characteristics, this strain could only be identified as Streptomyces sp.. On clavan, this strain displays good growth (17.5 g DCW/l after 96 h of cultivation) characterized by filamentous growth during the earlier days of cultivation followed by sporulation after 4 days. The flow behaviour of the Clavibacter broth was characterized, the fermentation culture broth behaves as a pseudoplastic fluid. The viscosity of the culture broth as well as of the purified clavan EPS, decreases when lyophilised supernatant of Streptomyces sp. YSDL-20 was added, indicating clavanase action. The viscosity decreases by 26% when the Clavibacter culture broth was incubated during 18 h with the crude Streptomyces enzyme source, whereas a 82% viscosity drop was observed, when the purified clavan EPS (10 g/l) was incubated with the lyophilised Streptomyces supernatant for 5 h.

Biotransformation↗
PubMed2002

Optimization of exopolysaccharide production by Tremella mesenterica NRRL Y-6158 through implementation of fed-batch fermentation.

In liquid culture conditions, the yeast-like fungus Tremella mesenterica occurs in the yeast state and synthesizes an exopolysaccharide (EPS) capsule, which is eventually released into the culture fluid. It is composed of an alpha-1,3-D-mannan backbone, to which beta-1,2 side chains are attached, consisting of D-xylose and D-glucuronic acid. Potato dextrose broth (PDB) seemed to be an excellent medium for both growth of the yeast cells and synthesis of the EPS. This medium is composed solely of an extract of potatoes to which glucose was added. Yet an important disadvantage of this production medium is the presence of starch in the potato extract, since Tremella cells are not capable of metabolizing this component; furthermore, it coprecipitates upon isolation of the polymer [3]. In this respect, it was essential to remove the starch in order to achieve high polysaccharide production and recovery. A good method was the removal of starch through ultrafiltration of the PDB medium before inoculation of the strain. This resulted in an excellent starch-free medium in which other components essential for polysaccharide production were still present [3]. Through implementation of single and cyclic fed-batch fermentations with glucose feed, 1.6- and 2.2-fold increases in EPS yield were obtained, respectively. Lowering the carbon source level by using a cyclic fed-batch technique might decrease the osmotic effect of glucose or any catabolite regulation possibly exerted by this sugar on enzymes involved in EPS synthesis.

Basidiomycota↗
PubMed2001

Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1.

AIMS: Optimization of the growth of Pediococcus damnosus NCFB 1832 and the production of pediocin PD-1 by traditional fermentation methods. METHODS AND RESULTS: Fermentation studies were conducted in De Man Rogosa and Sharpe (MRS) broth (Oxoid), preadjusted to specific pH values, and in MRS broth supplemented with various nitrogen sources, MnSO4, MgSO4 and Tween 80. The production of pediocin PD-1 closely followed the growth curve of Ped. damnosus NCFB 1832. Maximum levels of bacteriocin activity (3249 AU ml(-1)/O.D.max) were recorded in MRS broth with an initial pH of 6.7. In media with an initial pH of 4.5 bacteriocin activity as low as 222 AU ml(-1)/O.D.max was recorded. The highest bacteriocin activity was recorded in growth conditions allowing the greatest pH variation (highest DeltapH). The addition of bacteriological peptone (1.7%, w/v), MnSO4 (0.014%, w/v) and Tween 80 (3%, v/v) to MRS and adjustment of the medium pH to 6.7 resulted in a further increase in activity (from 3249 to 5078 AU ml(-1)/O.D.max). The same medium, but with an initial pH of 6.2, resulted in an 82.5% decrease in bacteriocin activity. CONCLUSIONS: Pediocin PD-1 production is not only stimulated by the presence of specific growth factors (e.g., bacteriological peptone, MnSO4 or Tween 80), but may also be stimulated by the lowering in pH during growth (highest DeltapH), and thus also the amount of organic acids produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of pediocin PD-1 by the wild-type producer strain was significantly improved by using a defined medium and traditional fermentation methods.

Anti-Bacterial Agents↗
PubMed2001

Microbial aerobic conversion of grass cell wall polysaccharides: screening and strain characterization.

A screening for microorganisms able to metabolize insoluble plant cell wall constituents was performed. As plant material, grass (Poaceae) was chosen. The selectivity of the screening medium was increased by previously extracting soluble sugars and proteins with 1 M phosphate buffer, pH 7. About 45 bacterial and 44 fungal strains were isolated. A gravimetrical method had to be optimized to determine microbial growth and the grass cell wall material converted, since conventional methods couldn't be applied due to interference of the grass particles. The selected fungi are the most promising group for the degradation of insoluble plant cell wall polymers; up till 30% conversion within 7 days was observed with the best fungal strains.

Aerobiosis↗
PubMed2001

Three-factor response surface optimization of the production of dextran dextrinase by Gluconobacter oxydans.

Response surface methodology (RSM) and a five-level three-factor central composite rotatable design (CCRD) were used to evaluate the effect of glycerol and peptone concentration and initial pH on dextran dextrinase (DDase) production by Gluconobacter oxydans. Optimal fermentation conditions were 20.59 g/l of glycerol, 6.67 g/l of mycological peptone and an initial pH of 6.14. The predicted DDase yield of the optimised fermentation was 0.207 U/ml, whereas an actual experimental yield of 0.208 +/- 0.025 U/ml was obtained.

Fermentation↗
PubMed2001

Metabolic engineering of Escherichia coli: construction and characterization of a gltA (citrate synthase) knockout mutant.

E. coli is one of the most important host organisms for recombinant protein production. However, growth and recombinant protein production can be limited by acetate accumulation during high-cell-density fermentations. Some of the strategies used to overcome this problem are based on the alteration of the genotype of the host. This paper discusses the construction and characterization of an E. coli gltA- knockout mutant. The knockout of the gene was confirmed by the loss of citrate synthase activity in an enzyme assay. Also the growth rate of the mutant on Luria Broth and Luria Broth + acetate was reduced.

Citrate (si)-Synthase↗
PubMed2001

Streptomyces strains as a source of enzymes, able to hydrolyse the Clavibacter exopolymer, clavan.

Two soil isolates, identified as Streptomyces sp. and designated as Streptomyces sp. YSDL-10 and Streptomyces sp. YSDL-20, were among the few microorganisms and able to hydrolyse and grow on clavan as sole carbon source. This biopolymer, very rich in L-fucose, is produced in by Clavibacter michiganensis subsp. michiganensis LMG 5604. The enzyme activity tentatively called clavanase was mainly detected in the culture liquid. However, qualitative tests showed that this activity was very weak in the supernatant. In order to test whether this activity was dependent of induction, several homo- and heteropolysaccharides as well as clavan were used as sole carbon source: the strains were grown on 10 g/l clavan, starch, cellulose, pectin, polygalacturonic acid, xylan or starch. After 5 days of growth, samples were centrifuged (10.000 rpm, 20 min) and the supernatants were lyophilised This lyophilised preparation was used as crude enzyme source. This preparation was dissolved in 500 microl phosphate buffer (pH 7.0) and added to 500 microl of clavan (0.5% w/v). Upon incubation of this mixture at 30 degrees C during 30 minutes, reducing sugars and L-fucose levels were quantified. The clavanase activity was relatively high in terms of reducing sugar content, but was weak in terms of L-fucose liberated. Nevertheless, qualitative tests performed on solid screening clavan medium showed large clearing zones around Streptomyces colonies. This indicates that clavan is split into oligosaccharides with sugar reducing moieties. The glycosidase activities detected in Czapek Dox-clavan medium were also weak, except for beta-D-xylosidase. This suggests that this enzyme activity could have some role in the clavanase enzyme complex.

Biomass↗