Influence of the rapeseed protein hydrolysis process on CHO cell growth.

Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media.

Improvement of Sporobolomyces ruberrimus carotenoids production by the use of raw glycerol.

The red yeast Sporobolomyces ruberrimus H110 was able to use glycerol as a carbon source. The highest concentration (0.51gL(-1)) and productivity (0.0064gL(-1)h(-1)) of carotenoids were achieved when raw glycerol from biodiesel production, containing around 1gL(-1) of fatty acids, was used as the carbon source, which represented increases of 27% and 1.5×, respectively, in relation to pure glycerol. Mass spectrometry analysis led to the identification of four carotenoids in the fermented samples, torularhodin, torulene, ß-carotene and γ-carotene. The use of raw glycerol also enhanced the proportion of torularhodin (69% against 59% in pure glycerol). The addition of individual fatty acids (palmitic, stearic, oleic and linoleic acids) to pure glycerol resulted in increases between 15% and 25% in maximum concentration and between 1.6× and 2.0× in productivity of carotenoids. The presence of palmitic and oleic acids increased the torularhodin proportion to 66%.

Production of a membrane-bound protein, the human gamma-glutamyl transferase, by CHO cells cultivated on microcarriers, in aggregates and in suspension.

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h-1, the highest cell density (near 1.3 x 10(6) cells ml-1), and the highest enzyme activity around 300 mU ml-1, which corresponded to a specific cellular level of 20 mU 10(-5) cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.

Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats.

Cell growth, lipid accumulation and cellular lipid composition of Yarrowia lipolytica growing on mixtures of industrial fats containing stearic, oleic, linoleic and palmitic acid have been studied. During growth, the strain incorporated oleic and linoleic acids more rapidly than the saturated fatty acids. Relatively high lipid accumulation (up to 0.44 g of lipids per g of dry matter) was observed when stearic acid was included in the culture medium. In contrast, substrates rich in oleic acid did not favor cellular lipid accumulation. The accumulated lipids, mainly composed of triacylglycerols (45-55% w/w), demonstrated a different total fatty acid composition compared with that of the substrate; in all cases, the microorganism showed the unusual capacity to increase its cellular stearic acid level, even if this fatty acid was not found in high concentrations in the substrate. This permitted the synthesis of interesting lipid profiles with high percentages of stearic acid and non-negligible percentages of palmitic and oleic acid, with a composition resembling that of cocoa-butter.

Induction of recombinant human gamma-glutamyl transferase by sodium butyrate in transfected V79 and CHO Chinese hamster cells.

Sodium butyrate was used to enhance biosynthesis rates of recombinant human gamma-glutamyl transferase (GGT) expressed under the control of the SV40 or the cytomegalovirus immediate early promoter, respectively, in transfected V79 and CHO Chinese hamster cell lines. Maximal induction of GGT specific activity in butyrate-treated cells ranged from 3 to 5-fold and resulted from a strong increase in the GGT mRNA ratio. We also observed that maximal transcription level in V79 cells occurred within 12 hr of treatment, whilst the cell proliferation was transiently arrested. Despite its processing requirements, induced GGT exhibited unchanged catalytic and physico-chemical features relative to human serum or hepatoma enzyme, thus appearing as an excellent model for further studies on human GGT.

Yarrowia lipolytica as a potential producer of citric acid from raw glycerol.

AIMS: To study the biochemical response of Yarrowia lipolytica LGAM S(7)1 during growth on raw glycerol (the main by-product of bio-diesel production units) in order to produce metabolic products of industrial significance. METHODS AND RESULTS: Yarrowia lipolytica was cultivated on raw glycerol or glucose in flasks. Although nitrogen-limited media were employed, growth was not followed by production of reserve lipid. Nitrogen limitation led to citric acid excretion. Growth and citric acid production parameters on glycerol were similar to those obtained on glucose. When high initial glycerol media were used, citric acid up to 35 g l(-1) (yield 0.42-0.44 g acid g(-1) glycerol consumed) was produced. CONCLUSIONS: Raw glycerol was an adequate substrate for Y. lipolytica. Growth was not followed by reserve lipid accumulation, but amounts of citric acid were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Raw glycerol is an industrial feedstock appearing in increasing quantities as the main by-product of bio-diesel production facilities. The present study describes an alternative way of glycerol valorization, with the production of remarkable amounts of citric acid, in addition to its main valorization way (production of 1,3-propanediol by bacteria).

Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures.

The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.

Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth.

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special((R)) enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N((R)) enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.