A low molecular weight alkaline proteinase fromConidiobolus coronatus

Summary An extracellular, low molecular weight alkaline proteinase (alkaline proteinase B) has been purified to homogeneity from the culture filtrate ofConidiobolus coronatus (NCIM 1238). A 12-fold purification was achieved with a specific activity of 29,760 u/mg. The enzyme had an optimum pH and temperature of 9.7 and 45°C respectively. It was most active towards casein and had a molecular weight of 6,800, the lowest reported so far. It was stable between pH 6.5–7.5. Alkaline proteinase B is a serine proteinase. It showed an esterolytic activity on N-benzoyl-L-tyrosine ethyl ester (BTEE) and was successfully used to resolve the racemic mixture of D, L-phenylalanine and D,L-phenylglycine and can thus potentially replace subtilisin Carlsberg in resolving the racemic mixture of amino acids.     

Production of an alkaline proteinase fromConidiobolus coronatus and its use to resolvedl-phenylalanine anddl-phenylglycine

An isolate ofConidiobolus coronatus (NCIM 1238) showed high proteinase activity (20.1 U/ml) and productivity (600 U/l.h) when 1% (w/v) glucose or sucrose was used as the carbon source in shake flasks. Addition of organic nitrogen sources, casein (2%), soybean flour (4%), liver extract (2%) or Edamin-S (2%), enhanced growth and proteinase production up to three-fold and seven-fold, respectively. The system was successfully run up to 6 l in a laboratory fermenter with a productivity of 600 U/l.h. The proteinase was successfully used to resolve the recemic mixtures ofdl-phenylalanine anddl-phenylglycine and thus could replace the currently used subtilisin.     

Oxygen transfer in animal cell culture medium

Both k(L)a and k(L) measurements were carried out by an unsteady state technique at impeller speeds ranging from 1.6 to 5.8 s(-1) in a mechanically agitated animal cell culture vessel of working volume 1.5 L. Checks were made that the time constant of the oxygen electrode was negligible compared to the time for aeration and that the oxygen electrode reading was not a function of agitator speed in the range employed. The k(L) values by surface aeration of (1.18-3.54) x 10(-5) m/s and k(L)a values by sparged aeration of (2.8-8.5) x 10(-4) s(-1) were found. The former are in reasonable agreement with published experimental values and the latter in accord with values estimated from published correlations based on agitator power input and aeration rate. The fluids used were water, basal medium, and basal medium supplemented with 5% (v/v) foetal calf serum; for each of these, k(L) and k(L)a values were similar. However, the addition of silicone antifoam (6 PPM) reduced the k(L)a value by ca. 50%.     

Effects of microcarrier concentration in animal cell culture

Results are presented which show how the microcarrier concentration affects the hydrodynamic environment in animal cell bioreactors. At low levels of agitation, no physical effects of microcarrier concentration were found. However, cell growth was strongly influenced by cell concentration. At high levels of agitation, a strong detrimental effect of microcarrier concentration was found. A new mechanism of hydrodynamic damage was identified which is second order in microcarrier concentration. The identification of this mechanism adds to the fundamental understanding of hydrodynamic phenomena in microcarrier bioreactors.     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus. Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administering APV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.     

Viscous reduction of turbulent damage in animal cell culture

Animal cells are exposed to turbulent fluid flow in many cell culture processes. If the turbulence in the flow is sufficiently strong, the cells will be damaged or killed by fluid-mechanical forces. Through an increase in viscosity, the turbulence can be damped and the hydro-dynamic damage can be reduced. In this article, new experimental results are presented which illustrate the protective effect of thickening agents. The results follow the prediction of a model based on Kolmogorov's theory of universal equilibrium in turbulent flow fields.     

Colorimetric pH measurement of animal cell culture media

Most animal cell culture media can be buffered using bicarbonate and high pressure CO₂ in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO₂ pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.     

Simulation and dynamic optimisation of animal cell culture

In animal cell culture, there are some 25 substrates that both have a significant effect on the culture performance and which can be measured with relative ease. A detailed dynamic simulation for such a culture has been produced and an optimisation policy that use this model to identify ideal media conditions has been developed. This paper describes an extension of that work to include the dynamic optimisation of cultures under fed-batch operation. Two different types of feeding policy were considered – in the first, discrete shots of feed were supplied, while in the second, feed was added continuously. Both policies offered significant improvements in the predicted productivity of the culture - up to 30% that of an experimentally optimisedbatch culture.     

Lethal events during gas sparging in animal cell culture

The lethal effects of gas sparging on hybridoma cells obtained from a chemostat culture were examined in a bubble column. Experiments were performed to identify and quantify the main hazardous event: bubble formation, bubble rising, or bubble breakup. The results indicate that bubble breakup is the main cause of cell death. The protective activity of the surfactant Pluronic F68 against sparging seems to result from a direct interaction with the cells rather than influencing bubble-liquid interface properties.     

Oxygen transfer properties of bubbles in animal cell culture media

Oxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles ( approximately 5 mm diameter) < intermediate bubbles ( approximately 1 mm diameter) < micron-sized bubbles ( approximately 100 mum diameter). Under certain conditions, the micron-sized bubbles were capable of achieving oxygen transfer rate up to 100 h(-1), a 10-20-fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new-born calf serum (NBCS) and/or Pluronic F-68 supplemented media. For the intermediate and micron-sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F-68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron-sized bubbles in Pluronic F-68 supplemented media containing antifoam agent and NBCS.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Selective removal of ammonia from animal cell culture broth

Serum-free perfusion cultures of hybridoma TO-405 cells were carried out in spinner flasks coupled with zeolite A-3 packed beads. Ammonia was selectively removed from the culture broth by passing cell free permeate from ceramic cross flow filtration, through the zeolite packed bed. Ammonia concentration in the culture broth was effectively maintained between 1 to 4 mmol/l which was below the inhibitory concentration for cell growth. Maximum cell density levels of 10⁷ cells/ml as well as improved percentage cell viability higher than in serum-supplemented cultures were feasible in this system.The possible effects of shear stress, generated by variation of the flow rates of the broth through the ceramic filter module, on the growth of the hybridoma cells were investigated. Backwashing, by reversing the direction of the permeate, was found necessary to prolong the life of the filter. Variation of the flow rates of the broth through the ceramic module between 0.29 m/s to 0.59 m/s did not cause immediate cell damage but growth was repressed at the higher flow rate.This study also showed that glutamine appears to be one of the factors limiting the growth of the hybridoma cells.