Improved performance in viscous mycelial fermentations by agitator retrofitting

For viscous mycelial fermentations it was demonstrated at the pilot-plant scale that the replacement of standard radial flow Rushton turbines with larger diameter axial-flow Prochem hydrofoil impellers significantly improved oxygen transfer efficiency. It was also determined that the Streptomyces broth under evaluation is highly shear thinning. Separate experiments using a Norcardia broth with similar Theological properties demonstrated that the oxygen transfer coefficient, K(L)a, can be greatly increased by use of water additions to reduce broth viscosity. These observations are consistent with the hypothesis that the improvement in oxygen transfer by changing agitator types is primarily due to an improvement in bulk mixing. A model is presented, based on the concepts of Bajpai and Reuss, which explains this improvement in performance in terms of enlargement of the well mixed micromixer region for viscous mycelial broths.     

On the mechanism of amplification of satellite II DNA sequences of the domestic goat Capra hircus

A restriction enzyme analysis of the satellite II DNAs of the domestic goat Capra hircus, sheep Ovis aries and ox Bos taurus (p = 1.720, 1.723 and 1.722 g/cm3, respectively) has been carried out and shows that, although all three are composed of repeat units of 700 base-pairs, goat satellite II is present in the genome primarily in the form of 2100 base-pair trimers. Unequal crossing-over between repeat units has produced an oligomer series, whose oligomers gradually decrease in copy number the further they are in molecular weight from the trimer. The trimer population is much more uniform than the monomer population, as most trimers have similar restriction patterns, whereas their component monomers differ considerably in their restriction properties. This heterogeneity was confirmed by cross-hybridization of the different monomers of a cloned trimer. Here, heterologous hybrids were much less stable than the homologous hybrids. Attempts were made to simulate such an oligomer series by computer, using a longitudinal (saltatory), and two horizontal (unequal crossover and drive) models. Simulations of both the saltatory and drive mechanisms could produce the oligomer series in approximately the observed ratios, but only the former could simultaneously produce other restriction properties of this sequence family. This was because the other restriction sites were in a different (monomer) register, and it is difficult for a drive model promoting traits in only one register to fix properties in different registers. The unequal crossover model proposed by Smith (1973, 1976) generally produced homogeneous arrays from heterogeneous arrays, but higher-order repeat structures could be produced when the efficiency of crossing-over between different monomer types was reduced. In most of these arrays, the dimer was the predominant oligomer, but in approximately 10% the trimer was predominant. Since the unequal crossover model produces dimeric arrays with high frequency given appropriate conditions, it is an attractive model for explaining the production of satellite DNAs whose structure has evolved through a series of doublings, such as mouse major satellite DNA and the "alphoid" satellite sequences of primates. Other factors necessary for this model to work are generally considered to be natural components of the speciation process. It is therefore suggested that, although the saltatory model conforms most closely to the observed structure of goat satellite II, this particular satellite DNA may represent one of the few cases when the unequal crossover mechanism does not give rise to a dimeric structure.     

The respiration of insects at reduced pressures II. The uptake of oxygen by Tenebroides mauritanicus

The respiration of larvae of Tenebroides mauritanicus exposed to reduced pressures in air was measured in a specially designed chamber by means of gas chromatography. Both O₂ uptake and CO₂ output were progressively reduced as the pressure was lowered from 200 to 35 mm Hg. There were differences in the amounts of gases exchanged when the insects were exposed under dry or moist conditions. When expired CO₂ was allowed to accumulate, in some experiments it had a marked stimulating effect on O₂ uptake between 60 and 200 mm. The respiratory quotient at the various pressures was calculated and the values found under different conditions of pressure and moisture are discussed.

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Zusammenfassung In der ersten Veröffentlichung dieser Reihe beschrieben Monro, Dumas und Buckland (1962) die Kohlendioxyd-Ausatmung der Larven von T. mauritanicus bei verringertem atmosphärischen Druck. Unter 65 mm nahm die CO₂-Ausatmung deutlich und fortschreitend ab. Um zu zeigen, daß dieser Verringerung der CO₂-Produktion tatsächlich eine Abnahme der Atmung entspricht, wurde ein Apparat entwickelt, mit dessen Hilfe O₂-Aufnahme und CO₂-Abgabe der Insekten gaschromatographisch gemessen werden konnten.Bei dreistündiger Exposition wurde unter den experimentellen Bedingungen gefunden, daß sich im Vergleich mit der Reaktion bei höherem Druck sowohl O₂-Aufnahme als auch CO₂-Abgabe bei 100, 60 und 30 mm fortschreitend verringert. Bei so niedrigem Druck war die Atmung während der Expositionszeit gleichmäßiger als bei höherem Druck, bei dem von Zeit zu Zeit deutliche Schwankungen auftraten. Bemerkenswert war ein deutlicher Unterschied in der Respirationsrate bei feuchtigkeitsgesättigter Luft und unter trockenen Bedingungen. Bei hoher Luftfeuchtigkeit war die O₂-Aufnahme zwischen 100 und 400 mm größer als bei Trockenheit und die CO₂-Abgabe war größer von 65 mm aufwärts. Daraus wird gefolgert, daß in diesem Druckbereich bei Trockenheit der Austausch beider Gase durch dauernden oder zeitweisen Verschluß der Atmungswege behindert wird.In einigen Versuchen wurde CO₂ durch Absorption aus der die Insekten umgebenden Atmosphäre entfernt. Abwesenheit von CO₂ verringerte die O₂-Aufnahme im Bereich von 60–200 mm besonders bei Feuchtigkeit. Bei Trockenheit war die O₂-Aufnahme bei hohem CO₂-Gehalt signifikant geringer. Hier mag wiederum Schließung der Atmungswege die stimulierende Wirkung des CO₂ vermindert haben.Berechnungen des Respirationsquotienten aus den verfügbaren Daten zeigen eine stetige Abnahme dieses Wertes mit abnehmendem Druck bei Feuchtigkeit, während der R.Q. in trockener Umgebung bei jedem Druck ziemlich konstant blieb. Es werden keine endgültigen Schlüsse aus diesen Berechnungen gezogen, aber es wird angenommen, daß die feuchte Umgebung einen freieren Gasaustausch durch die Atmungswege erlaubt und daher bessere Bedingungen zur Bestimmung des tatsächlichen R.Q.-Wertes bei vermindertem Druck bietet.

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In this paper pressures are given as absolute pressures expressed in mm Hg.     

Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region.

In-frame and frameshift mutations were introduced into the pre-S region (1,212 base pairs) of duck hepatitis B virus. The in-frame mutants retained the inserted 12 nucleotides, while the frameshift mutants either reverted to wild type or exhibited a 10-nucleotide compensatory deletion downstream of the original mutation site. Thus, although duck hepatitis B virus has a compact and highly economical genome organization, it can replicate despite alterations of up to 9 amino acid codons in the pre-S and P open reading frames.     

Identification of two amino acids in the hemagglutinin glycoprotein of measles virus (MV) that govern hemadsorption, HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains.

We have used site-directed mutagenesis of the hemagglutinin (H) glycoprotein of measles virus (MV) to investigate the molecular basis for the phenotypic differences observed between MV vaccine strains and recently isolated wild-type MV strains. The former downregulate CD46, the putative cellular receptor of MV, are positive for hemadsorption, and are fusogenic in HeLa cells, whereas the latter are negative for these phenotypic markers. CD46 downregulation in particular, could have profound consequences for the immunopathology of MV infection, as this molecule protects the cell from complement lysis. Mutagenesis of two amino acids, valine and tyrosine at positions 451 and 481, respectively, in the H protein from the vaccine-like Hallé MV strain to their counterparts, glutamate and asparagine, in the H protein from the wild-type Ma93F MV strain (creating the V451E/Y481N double mutation) abrogated CD46 downregulation, HeLa cell fusion, and hemadsorption. The converse double mutagenesis of the Ma93F H protein (E451V/N481Y) transferred the CD46-downregulating, fusogenic, and hemadsorption functions to this protein. The data provide the first mapping study of the functional domains of MV H. The consequences of these results for MV vaccine design and the role of CD46 in MV infection are discussed.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Distribution of soil invertase in relation to the root systems ofPicea sitchensis (Bong.) Carr. andAcer pseudoplatanus L. during development of young plants

A method is described for sampling rhizosphere soil under newly establishedPicea sitchensis andAcer pseudoplatanus. The technique involves taking soil samples to a depth of 150 mm at 100 mm intervals along transects, each 45° from its neighbour, radiating from the base of the stem. Invertase activities were measured in the soil samples and compared to their activities in fallow and rhizosphere soils. When the field soil was dry, the tree root systems were carefully excavated to retain as many fine roots as possible. The distribution of the soil invertase was matched to the spatial distribution of the roots showing the precise position of the rhizosphere relative to the initial blind soil sampling. Statistics were applied to derive equations for calculating the percentage enzyme activity relative to that found in rhizosphere soil at various locations radiating from the base of the stem. This information was subsequently applied to soil sampled under trees of the same age as those excavated to give a non-destructive method for sampling rhizosphere soil routinely from under a large number of trees.     

Development of a recovery and recycle process for a pseudomonas lipase used for large-scale enzymatic synthesis

Enzymes are potential catalysts for a wide range of large-scale chemical synthesis steps, particularly when the creation of a specific chiral center is desired. The efficient recycling of the enzyme catalyst and the removal of carryover impurities were crucial factors in the improvement of a stereoselective ester hydrolysis step used in the synthesis of a selective leukotriene antagonist. In this enzymatic reaction step, the substrate and product were both largely insoluble, while the enzyme was soluble in the aqueous reaction mixture. Microfiltration and ultrafiltration of the slurry reaction mother liquor indicated near 100% enzyme protein recovery, while activity recovery was about 70% to 80%. These activity losses might be accounted for by enzyme degradation (1 to 2 mg/L . h) during the 40-hour reaction period. Dissolved impurities, principally a diacid byproduct, in the enzyme recycling stream were reduced 60% to 70% by either lowering the solution pH to 4.0 or raising the solution ionic strength to 1 M. (c) 1993 John Wiley & Sons, Inc.     

Use of comparative reasoning tools for evaluation of the effect of sterilization conditions on fermentations to produce acidic fibroblast growth factor

The effects of various medium sterilization conditions on fermentations of a recombinant, acidic fibroblast growth factor (aFGF) producing Escherichia coli have been studied. Changes in the medium resulting from sterilization were monitored by pH and absorption spectra. This simple experiment provided excellent data for the demonstration of the usefulness of comparative reasoning tools in order to evaluate the effect of sterilization on fermentation performance. The time profiles of the main parameters (e.g., carbon dioxide evolution rate, dissolved oxygen, pH, and aFGF productivity) were simplified into piecewise contiguous linear segments, each of which was sequentially numbered. The length, position, and slope of each tine were then characterized. Application of the comparative reasoning tools confirmed that separate sterilization of the glucose was necessary for the success of the process, despite adding to the cost and complexity. The comparative data analysis also showed that scaleup with longer sterilization holding and cooling times would not be detrimental to aFGF production. (c) 1995 John Wiley & Sons, Inc.