Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities.

The TrxRl form of thioredoxin reductase (TrxR) was the major form of the enzyme isolated from HeLa cells grown in a fermentor at 35 degrees C under controlled aeration conditions favorable to growth, nominally 30% of saturation of dissolved oxygen or 8 ml of oxygen in a liter of medium. This TrxR1 form was not retained on a heparin affinity matrix, it contained one selenium per subunit, was highly active catalytically, and showed strong cross-reactivity with anti-rat liver TrxR1 polyclonal antibodies. At higher aeration, 50% of saturation of dissolved oxygen or 12 ml of oxygen in a liter of medium, HeLa cell growth was slower and additional TrxR forms that bound to heparin were present in purified enzyme preparations. A minor component, TrxR2, the mitochondrial form of TrxR, was detected in the heparin-bound enzyme fraction. One enzyme form that contained less selenium (ca. 0.5 Se per TrxR subunit) was only about 50% as active with thioredoxin or 5,5'dithiobis(2-nitrobenzoic acid) as substrate. Cross-reactivity of this form with anti-rat liver TrxR1 polyclonal antibodies was very weak. The isoelectric point of the low Se enzyme, 5.85, was higher than that, 5.2-5.4, of normal Se content enzyme. Affinity of purified fully active TrxR1 to heparin could be induced by reduction with NADPH or tris-(2-carboxyethyl)phosphine (TCEP). Under anaerobic conditions there was complete retention of Se indicating that an enzyme conformation change effected by reduction was involved. The TCEP-reduced enzyme form was very oxygen labile and upon exposure to air both the Se content and catalytic activity decreased by about 50%. Addition of millimolar concentrations of NADPH or NADP(+) to the TCEP-reduced enzyme gave full protection from oxygen inactivation. TrxR1 exhibited weak peroxidase activity with H(2)O(2) as substrate in the presence of an NADPH-generating system but this activity was unstable. Specific alkylation of the selenocysteine residue of TrxR1 which completely inhibits the NADPH-dependent reduction of disulfides also destroyed peroxidase activity.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Influence of environmental and nutritional factors in the production of astaxanthin from Haematococcus pluvialis

Astaxanthin extracted from green algae is desirable in the food and pharmaceutical industries due to its antioxidant properties. The green unicellular clear water microalga Haematococcus pluvialis has a high production rate of astaxanthin; indeed, it contains more than 80% astaxanthin content in its cells. This remarkable astaxanthin production is commonly obtained under stress conditions such as nutrient deficiency (N or P), high NaCl concentrations, variations of temperature, and other factors. In this vein, a great research effort has been oriented to determine optimal conditions for astaxanthin production by H. pluvialis.The objective of the present study was the analysis of environmental factors, such as light intensity, aeration and nutrients on the growth and astaxanthin production of H. pluvialis. Maximum growth of H. pluvialis obtained was 3.5x10(5) cells/ml in BBM medium at 28 degrees C under continuous illumination (177 micromol photon m(-2)s(-1)) of white fluorescent light, with continuous aeration (1.5 v.v.m.). Meanwhile, maximal astaxanthin production was 98 mg/g biomass in BAR medium with continuous illumination (345 micromol photon m(-2)s(-1)), with 1 g/l of sodium acetate and without aeration.     

The Aeration of Catharanthus roseus L. G. Don Suspension Cultures in Airlift Bioreactors: The Inhibitory Effect at High Aeration Rates on Culture Growth

An investigation was carried out to examine the effect of aerationon the growth of Catharanthus roseus suspension cultures inairlift bioreactors. A high aeration rate (0·86 v.v.m.)was found to inhibit the growth of cultures. Venting culturesat a high rate with low oxygen content gas mixtures was equallyinhibitory to culture growth, showing that high aeration wasnot inhibitory as a result of oxygen toxicity. The dissolvedcarbon dioxide tension was found to be lower in cultures operatedat high aeration than those operated at low aeration. Supplyingexogenous CO₂ to cultures at high aeration restored the CO₂tension to values normally encountered at a low aeration rate,and was found to alleviate the inhibitory effects at high aeration.However, further increasing the CO₂ supply to cultures was foundto be severely inhibitory to growth. Therefore, the growth ofC. roseus cultures is very sensitive to dissolved CO₂ concentration,growth being inhibited at values either higher or lower thanan optimum. Key words: Aeration, carbon dioxide, Catharanthus roseus suspension culture     

The effect of agitation and aeration on the synthesis and molecular weight of gellan in batch cultures of Sphingomonas paucimobilis

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymer synthesis.Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). An increase in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis.Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions.The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods.While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecular weight (at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research.     

Relationship Between the Sterol Content of Yeast Cells and Their Fermentation Activity in Grape Must

In grape must of high sugar concentration, yeast growth, the viability rate of “resting” yeast cells, and fermentation activity were stimulated under certain conditions of aeration and temperature. This stimulation might be interpreted as being a result of the yeast cell sterol content. The addition of certain sterols to the fermenting medium was able to increase this sterol content. According to aeration conditions of the medium, which determined the sterol content of yeasts, the sterols added in the medium acted as (i) growth factors, (ii) fermentation inhibitors, and (iii) survival factors for the yeast.     

Operation conditions affecting the performance of airlift reactors for immobilized enzyme reactions

Summary The applicability of concentric airlift reactors for immobilized enzyme reaction has been studied. Because of simple mechanical structure, good mixing, relatively low shear stress, and lower power consumption, the airlift reactor is apealing for immobilized enzyme reaction. The operation conditions can be optimized by manipulating aeration rate, top clearance space, and solid content.     

Induced synchrony in Cryptococcus neoformans after release from G2-arrest

Cryptococcus neoformans was grown first to OD 4 under moderate aeration, then diluted 2.5 times with fresh medium, and grown under limited aeration for 5 h. Oxygen concentration decreased from 5-6 mg l(-1) to 1.5 mg l(-1) 1 h after the shift to limited aeration, and remained at a similar level thereafter. In all the eleven strains examined the shift caused unbudded G(2)-arrest in more than half of the cells. In three strains more than 80% of the cells were arrested in unbudded G(2), and, therefore they were selected for synchrony experiments. After being shifted to extensive aeration again, the cells resumed growth by synchronous budding, followed by synchronous nuclear division. This method has turned out to be a good tool to prepare synchronized culture in C. neoformans, especially when a large amount of synchronized cells is needed. This is worthy of attention, since synchronous cultures after release from G(2)-arrest have not been reported yet in any yeast species.     

中华猕猴桃良种选育研究

本文扼要叙述了中华猕猴桃原产地的生态条件,分析了引入栽培园中所遇到的突出问题,提出了中华猕猴桃从高海拔山区引入低海拔丘陵平原应解决的关键技术,初步总结了在武汉风土条件下的栽培经验,简述了优株选育途径和效果,介绍了武植3号等优良品系的主要经济性状。     

Regulation of Glutamine Synthetase X. Effect of Growth Conditions on the Susceptibility of Escherichia coli Glutamine Synthetase to Feedback Inhibition

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).     

Fat formation in preformed fungal mats of penicillium notatum

A fat-forming fungus:Penicillium notatum was grown on a physiologically balanced medium for 4 days. The old medium was then replaced by a fat-promoting medium and the fungal cultures were reincubated for a further period of 10 days. Active growth was resumed. Protein synthesis also continued actively at the expense of the low nitrogen supply in the replacement medium and soon stopped when proteolysis started. Fat formation, on the other hand, started and proceeded for a longer period at a very high rate leading to a very high fat yield.When the fungal mats were replaced on the fat-promoting medium at different nitrogen concentrations, the medium with the lowest nitrogen supply gave rise to weak growth and low fat yield as well. The highest nitrogen supply gave rise to active growth with high protein content and low fat yield. At a certain concentration of nitrogen, there was a high growth rate involving the highest fat yield. It is concluded that replacement medium deficient in nitrogen is not the most favorable for fat formation. The nitrogen level in the replacement medium should be adjusted so as to afford good growth and consequently good utilization of excess carbohydrates in fat syntheses.     

Relationship between the quality of the inoculum material and carminomycin biosynthesis by a culture of Actinomadura carminata]

The effect of the inoculum mycelium quality on carminomycin biosynthesis by Actinomadura carminata was studied. The time of the organism growth on the culture medium containing cornsteep liquor continued for 6 hours without losing by the inoculum of its seeding qualities during that period. The mycelium growth in the inoculum was more intensive under conditions of moderate aeration, i.e. 0.98-2.64 mg O2H1-min. Anincrease in the aeration rate up to 18.56 mg O2/1-min resulted in the growth suppression up to 40 per cent. No correlation between the aeration rate during the inoculum growth and the culture capacity for carminomycin biosynthesis and of the content of the complex in active components the fermentation medium were observed, when a 5-10 per cent of inoculum was used.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

Nitrate Reductase and Soluble Cytochrome c in Spirillum itersonii

The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.     

A 24-microwell plate with improved mixing and scalable performance for high throughput cell cultures

Multi-well plates are widely used in high throughput drug screening, cell clone development, media design and cell culture optimization in the biotechnology industry. The reproducibility and data quality of cell cultures in multi-well plates are greatly affected by mixing, aeration, and evaporation. A novel 24-microwell plate (MWP) with static mixers for improved mixing and aeration, and gas permeable lids for reduced evaporation was developed for cell cultures. Mixing, oxygen transfer, evaporation, and cell proliferation as affected by the static mixer, shape of the well and agitation rate were studied. The static mixer improved mixing pattern and reduced cell aggregation under orbital shaking conditions. Consequently, the static mixer also improved cell proliferation with a significantly higher specific growth rate in round wells. In general, consistent growth kinetics was observed for cells cultured on the plate. Overall, the MWP improved the data quality with smaller standard deviations and better reproducibility. Furthermore, CHO cells cultured in the MWP gave similar kinetics in glucose consumption, lactate production, cell growth and viability, and antibody production in a serum-free medium to those cultured in spinner flasks, demonstrating its scalable performance and potential application in high throughput screening for cell culture process development.     

Regulation of the biosynthesis of aminoacylase and pigments by strains of Actinomyces of the genus Streptoverticillium

For using actinomyceteous extracellular aminoacylase in production of optically active amino acids it is necessary to have the fermentation broth with lowered contents of extracellular pigments whose accumulation dynamics in the process of the strain growth correlates with the dynamics of the enzyme biosynthesis. The results of the studies showed that it was possible to regulate biosynthesis of the aminoacylase and pigments in the direction of increasing the enzyme production and decreasing the pigment formation by using a medium of the respective composition and providing the respective high temperature and sufficient aeration for the strains.     

Growth control and ppGpp synthesis in Brevibacterium flavum cells at various medium mixing rates and aeration intensities

Under the conditions of various aeration and medium mixing intensities (even at a constant medium oxygen partial pressure value), the variations in the RNA synthesis as well as the growth rate of lysine producing Brevibacterium flavum strains are inversely correlated with the ppGpp concentration in the cells. An increase in the ppGpp synthesis and a decrease in the RNA content in the cells was observed in the cases of a low cell energy charge value (lower than 0.6). This took place in the cases of bacterial cultivation at a low or very high medium aeration and mixing intensity. Hence, the energy production in the cells, the ppGpp synthesis and the growth control mechanism in Corynebacteria may be regarded as connected processes.     

Biologische Erfahrung mit einem blasenfreien Begasungssystem (CHEMCELL)

The increased interest in large scale production of biologically active molecules, as for example monoclonal antibodys, hormones, proteins and enzymes, has stimulated a rapid development of different methods to cultivate eukaryotic cells. Further progress in this field of modern biotechnology is expected not only from the selection of more productive cell lines and more efficient cultivation techniques, but also from the improvement in new bioreactor design and operation, which guarantees increased productivity per unit volume and reduces the downstream processing. Most important factors for a new reactor design in future will be the energy- and mass transfer, shear stress and scaleability. The provision of an adequate oxygen supply to large scale reactors is the most critical barrier to scale up. If oxygen is limited in a small degree, the result is inhibition of cell density and cellular efficiency in the production of the desired biomolecules. In addition, when methodologies are used which allow high cell densities and high metabolic active cells, the oxygen transfer becomes more important and, at the same time, more difficult. Since direct sparging of air into the cell-containing medium causes problems such as shear forces and foaming, new, efficient methods in bubble free aeration must be utilized. A new aeration system is presented, which is suitable to bubble-free aeration as well to separation of microcarrier-anchered cells from the harvested medium when running in a continuous perfusion mode. The efficiency of the CHEMCELL System regarding aeration and cell retention is demonstrated by the growth kinetics of BHK 21-cells in batch and perfusion mode.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Oxygenation of intensive cell-culture system

The abilities of various methods of oxygenation to meet the demands of high-cell-density culture were investigated using a spin filter perfusion system in a bench-top bioreactor. Oxygen demand at high cell density could not be met by sparging with air inside a spin filter (oxygen transfer values in this condition were comparable with those for surface aeration). Sparging with air outside a spin filter gave adequate oxygen transfer for the support of cell concentrations above 10⁷ ml^–1 in fully aerobic conditions but the addition of antifoam to control foaming caused blockage of the spinfilter mesh. Bubble-free aeration through immersed silicone tubing with pure oxygen gave similar oxygen transfer rates to that of sparging with air but without the problems of bubble damage and fouling of the spin filter. A supra-optimal level of dissolved oxygen (478% air saturation) inhibited cell growth. However, cells could recover from this stress and reach high density after reduction of the dissolved oxygen level to 50% air saturation.