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Biomedical subjects

M Moo-Young

Publications and source records attributed to M Moo-Young.

At least 19 recordsLinked to original sources
PubMed2005

Medium optimization for hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods.

Statistics-based experimental design was used to investigate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl2.2H2O) on hen's egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2(5-1) fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other factors were not important within the levels tested. The method of steepest ascent was used to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g L-1, peptone 34 g L-1, ammonium sulfate 11.9 g L-1, yeast extract 0.5 g L-1, and CaCl2.2H2O 0.5 g L-1. This medium was projected to produce, theoretically, 212 mg L-1 lysozyme. Using this medium, an experimental maximum lysozyme concentration of 209+/-18 mg L-1 verified the applied methodology.

Algorithms↗
PubMed2005

Rheology and hydrodynamic properties of Tolypocladium inflatum fermentation broth and its simulation.

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.

Ascomycota↗
PubMed2004

Parameter oscillations in a very high gravity medium continuous ethanol fermentation and their attenuation on a multistage packed column bioreactor system.

The quasi-steady-states, marked by small fluctuations of residual glucose, ethanol, and biomass concentrations, and sustainable oscillations marked by big fluctuations of these monitored fermentation parameters were observed during the continuous ethanol fermentation of Saccharomyces cerevisiae when very high gravity media were fed and correspondingly high ethanol concentrations reached. A high ethanol concentration was shown to be one of the main factors that incited these oscillations, although the residual glucose level affected the patterns of these oscillations to some extent. The lag response of S. cerevisiae to high ethanol stress that causes the shifts of morphology, viability loss, and death of yeast cells is assumed to be one of the probable mechanisms behind these oscillations. It was predicted that the longer the delay of this response was, the longer the oscillation periods would be, which was validated by the experimental data and the comparison with the oscillatory behaviors reported for the ethanologen bacterium, Zymomonas mobilis. Furthermore, three tubular bioreactors in series were arranged to follow a stirred tank bioreactor to attenuate these oscillations. However, exaggerated oscillations were observed for the residual glucose, ethanol, and biomass concentrations measured in the broth from these tubular bioreactors. After the tubular reactors were packed with Intalox ceramic saddle packing, these oscillations were effectively attenuated and quasi-steady-states were observed during which there were very small fluctuations of residual glucose, ethanol, and biomass within the entire experimental run.

Apoptosis↗
PubMed2004

Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods.

A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified. The model was then used for optimizing the feeding schedule in fed-batch culture. To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements. Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results. Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation. The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation. Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable.

Algorithms↗
PubMed2004

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions.

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l(-1) glucose. An average ethanol concentration of 124.6 g l(-1) or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h(-1). The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.

Biomass↗
PubMed2002

Bioprocessing strategies for improving hen egg-white lysozyme (HEWL) production by recombinant Aspergillus niger HEWL WT-13-16.

Hen egg-white lysozyme (HEWL) production by recombinant Aspergillus niger HEWL WT-13-16 from a cDNA under the control of the A. niger glucoamylase promoter was used as a model system. The fungal mycelium was either immobilized on porous Celite 560 micro-carrier or grown in suspension as pelleted and dispersed forms. The objective was to reduce the protease activity that adversely affects the expressed HEWL. Free suspension culture at uncontrolled pH served as the benchmark. The control of pH during growth at pH 4.0 gave rise to a greater than five-fold reduction of protease activity in suspension culture. An additional 38.5% decrease in protease activity was achieved in mycelial-pellet cultures in comparison to a 40.9% decrease in protease activity obtained with Celite 560 beads in an airlift vessel at controlled pH. The specific HEWL yields were 5.8, 5.0 and 4.1 mg/g dry wt. for the free suspension, mycelial-pellet, and Celite-560-immobilized cultures, respectively.

Animals↗
PubMed2002

Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized Aspergillus niger cultures.

The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions. Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads. Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor. Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus. Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g(-1), while glucoamylase specific activity increased from 205 to 350 U g(-1). The effect was more pronounced in bioreactor cultures. A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor.

Aspergillus niger↗
PubMed2001

Peptidases affecting recombinant protein production by Streptomyces lividans.

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.

Aminopeptidases↗
PubMed2000

Single versus multiple bioreactor scale-up: economy for high-value products.

The economy of scaling-up a bioreactor by increasing the number of units was investigated with respect to an integrated flowsheet. For the production of t-PA from animal cells, a base case flowsheet using a single large bioreactor was compared to a multiple bioreactor case. Simulation of the complete flowsheets for the two cases showed that a multiple bioreactor approach to scale-up increases the return of investment (ROI) of the base process by 122%. This enormous increase in ROI results from the smaller size of the downstream units compared to the base case, since downstream processing accounts for about 80% of the total cost for high value products like t-PA. Proper scheduling of the downstream units allowed sharing of the equipment by the bioreactors. A breakdown of the equipment purchase cost showed that cost related to cell culture equipment increased from 14% for the base case to about 37% for the multiple bioreactor case. The contribution from chromatography columns to the total equipment purchase cost, on the other hand, decreased from 52 to 33%.

Journal Article↗
PubMed1997

Plasmid instability kinetics in continuous culture of a recombinant Saccharomyces cerevisiae in airlift bioreactor.

Plasmid instability of a recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) was examined during continuous culture in a nonselective medium in an airlift bioreactor. The recombinant strain contained a 2-micron based shuttle vector pGAC9 and expresses Aspergillus awatnori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter. The changes in the fraction of plasmid-bearing cells and glucoamylase activity followed first-order kinetics. Expressed as a function of time, the decay rates of both the plasmid-bearing cell fraction and glucoamylase expression increased with increasing dilution rates. If expressed as a function of cell generations, the decay rates were nearly constant over the dilution rates tested. The results indicated that the growth rate difference between plasmid-bearing and plasmid-free cells was negligible. This was probably due to the low copy number of the 2-micron based yeast shuttle vector. Thus the contribution of preferential growth to apparent plasmid instability was negligible. A novel numerical method is proposed to evaluate the parameters related to plasmid stability. The estimated values of probability of plasmid loss (P = 0.0499) were nearly constant at different dilution rates. No significant effect of growth rates on plasmid instability was observed. The proposed kinetics agreed well with experimental observations.

Bioreactors↗
PubMed1996

Bioprocessing with genetically modified and other organisms: case studies in processing constraints.

Whereas the gene stability related considerations are important in bioprocessing with recombinant cultures, bioreactor design and scale-up require attention to the often reduced shear tolerance of the genetically altered biocatalysts relative to the corresponding wild strains. In addition, the peculiarities of expression of the rDNA product impact the downstream recovery methods. As a consequence, a bioprocessing scheme and the process machinery designed for a naturally occurring organism may need significant modifications for use with a genetically modified variety of the same organism. The case studies described highlight some of the processing constraints and consideration of general relevance.

Animals↗
PubMed1996

Tissue-type plasminogen activator: characteristics, applications and production technology.

Plasminogen activators have immense clinical significance as thrombolytic agents for management of stroke and myocardial infarction. Tissue-type plasminogen activator (tPA) is generally preferred as being effective and safer than either urokinase or streptokinase type activators. Large-scale production of tPA became possible through groundbreaking developments in cell lines and bioprocess technology. Nevertheless, at thousands of dollars per treatment, tPA remains expensive. Enhancing cellular productivity and downstream product recovery through new approaches continue to be major challenges as discussed in this review. Recent clinical experience suggests the need for yet better fibrinolytic agents and attempts are underway to modify the tPA molecule to second generation products. Emerging trends in this field are outlined.

Journal Article↗
PubMed1996

Plasmid stability in recombinant Saccharomyces cerevisiae.

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.

Journal Article↗
PubMed1995

Effects of the hydrodynamic environment and shear protectants on survival of erythrocytes in suspension.

Survival of media-suspended porcine erythrocytes exposed to various hydrodynamic environments was investigated with and without such shear protectant additives as bovine serum albumin, dextran and the non-ionic surfactant Pluronic F68. Erythrocytes provided a model cell population with cells of a uniform size, metabolic state and shear tolerance. Because the cells were non-growing, any shear adaptation effects were avoided. Cell lysis was followed by microscopic counts or release of haemoglobin. The cells were susceptible to agitation damage in unaerated shake flasks agitated at 100 rpm or greater. Relative to additives-free operation, the presence of 0.1% (w/v) dextran or albumin prolonged cell survival, but Pluronic F68 actually enhanced cell lysis in flasks agitated at 100 rpm. The protective effect of the additives depended on the hydrodynamic conditions. The protective effect of albumin was demonstrated also in aerated conditions in a split-cylinder airlift bioreactor (aspect ratio of 8.8; riser-to-downcomer cross-sectional area ratio of 1.0; specific power input of 0.34 W m-3). Comparison of the cell lysis characteristics in the airlift device and the best case performance of the shake flask showed longer survival in the flask (100 rpm); however, the length of survival in the reactor (approx. 70 h) was sufficient for practical purposes. In all cases, the cell lysis pattern conformed initially to zero-order dependence in cell concentration, becoming first-order after varying degrees of exposure to hydrodynamic forces. Fatigue failure of cells was inferred.

Animals↗
PubMed1995

Isolation of a recombinant intracellular beta-galactosidase by ammonium sulfate fractionation of cell homogenates.

The Escherichia coli beta-galactosidase (EC 3.2.1.23) expressed intracellularly as soluble, biologically active enzyme in the yeast Saccharomyces cerevisiae was recovered from clarified homogenates of the yeast cells by precipitation with crystalline ammonium sulfate. Effects of salt saturation (0-80%) of the homogenate, the initial total protein level (2-20 g.L-1) and the processing pH (6-8) on the enzyme and protein recovery were investigated. As the ammonium sulfate concentration increased, the enzyme was precipitated preferentially and at 30% salt saturation nearly all had been recovered in the precipitate. In contrast, at this salt concentration only 25% of the total protein had precipitated. Preferential precipitation of beta-galactosidase was associated with the hydrophobic nature of this large protein. Complete precipitation of the total protein required salt concentrations exceeding 70% of saturation. The salt concentration needed for complete recovery of the enzyme was not sensitive to the processing pH. Over the salt saturation level of 20-50%, the enzyme precipitation followed the Cohn equation. The salting-out constant was strongly affected by the initial protein level in the homogenate; higher values were observed at lower protein concentrations. The salting-out constant was unaffected by the processing pH; however, the Cohn parameter B was pH dependent in addition to being affected by the initial protein concentration. Within the beta-galactosidase stability range of pH 6-8, pH variations alone (no added salt) proved ineffective in precipitating the enzyme.

Ammonium Sulfate↗
PubMed1994

Antimicrobial activity of bark extracts of Bridelia ferruginea (Euphorbiaceae).

Water and ethanol extracts of Bridelia ferruginea were examined for phytochemical and antimicrobial properties. The extracts, which were tested at a final concentration of 5 mg/ml, produced in vitro antimicrobial activities in assays against hospital strains of Staphylococcus aureus, Candida albicans, Staphylococcus epidermidis, Escherichia coli, Streptococcus lactis, Proteus vulgaris, Proteus mirabilis, Streptococcus pyogenes and Klebsiella sp. The zones of inhibition produced by the extracts in agar diffusion assays against the test micro-organisms ranged from 4 to 20 mm, while the chloramphenicol antibiotic control produced zones that measured 15-36 mm. Preliminary phytochemical analysis of the plant extracts showed the presence of phenols and tannins. Sesquiterpenes, anthroquinones, and saponins were not detected in the extracts. The Gram-negative bacteria appeared to be more susceptible (4-20 mm) to the antimicrobial effect of the extracts than the Gram-positive organisms (4-18 mm).

Anti-Bacterial Agents↗
PubMed1994

Disruption of a recombinant yeast for the release of beta-galactosidase.

A recombinant yeast, Saccharomyces cerevisiae, expressing Escherichia coli beta-galactosidase gene under the control of CYC1 constitutive promoter of the yeast, was disrupted in a continuous flow, high speed, bead mill for the release of intracellular beta-galactosidase (EC 3.2.1.23). Release of the beta-galactosidase activity was characterized with respect to glass bead loading in the grinding chamber (70-85% of chamber volume), diameter of the beads (0.25-0.75 mm), number of passes of the cell slurry through the mill (0-6 passes), flow rate of the slurry (25-250 mL.min-1), cell concentration in the slurry (5-20 gDW.L-1), the agitation rotor speed (1000-4000 rpm) and the pH of the slurry (pH 5-10). The optimal conditions for the release of the enzyme were pH 6.0-9.0, 85% loading of 0.5 mm diameter beads and an agitation speed of 2000 rpm. The enzyme release followed first-order kinetics. For otherwise fixed conditions, the extent of cell disruption increase with increasing bead load, number of passes and agitation rotor speed. Cell concentration did not affect disruption. The release of beta-galactosidase activity declined with increasing flow rate of the cell slurry through the mill, but the disruption rate constant increased with flow rate. Under optimal condition, three passes through the grinding chamber were sufficient to release all of the enzyme. In comparison with disruption in the bead mill, chloroform-sodium dodecyl sulfate induced lysis of cells was ineffective in releasing the enzyme quantitatively.

Biotechnology↗