Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

A high affinity interaction of plasminogen with fibrin is not essential for efficient activation by tissue-type plasminogen activator

Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1-77)), and Mini-Pg (lacking kringles 1-4) bound Fn with K(d) values of 3.1, 0.21, and 24.5 μm, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.     

吖啶酯发光法分析测定组织型纤溶酶原激活剂的研究

本文建立了灵敏度高、能定量测活且成本低廉的吖啶酯(AE)发光分析组织型纤维溶酶原激活剂(t-PA)的方法。该法灵敏度达0.125 IU/ml;在t-PA 0.125～8.0IU/ml范围内,t-PA活力单位对数与其发光计数呈良好的线性关系(r=0.997);批内变异系数为8％(n=8);回收率为86～122％。本法用于测定人黑色素瘤细胞mt-PA、CHO细胞表达的rt-PA,获得与纤维蛋白琼脂板测定法(FAPA)相一致的结果;测定健康人静脉闭塞前后血浆中t-PA活性的变化,显示出明显差别,静脉闭塞后血浆中t-PA活性约为静脉闭塞前的2～3倍。该法对肺癌患者血浆t-PA活性测定结果明显高于正常人,因而具有临床诊断价值。     

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium

Fourteen yeast strains were screened for production of 2-phenylethanol from l-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l^–1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l^–1 at 35 °C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.     

Stable production of an analog of human tissue plasminogen activator from culturedDrosophila cells

We have studied the expression of an analog of human tissue plasminogen activator, FK2P, inDrosophila Schneider 2 cells. A number of promoters were tested, including theDrosophila metallothionein promoter (MTd), baculovirus immediate early promoter (IE),Drosophila copia promoter, mouse metallothionein promoter, cytomegalovirus immediate early promoter with or without intron, SV40 immediate early promoter, and human elongation factor 1 promoter. Two of these promoters drove significant expression of FK2P. The MTd promoter is tightly regulated and upon induction with copper or cadmium expression of FK2P increases as much as 180-fold, accumulating in the culture medium to about 7 g FK2P/10⁶ cells/day as determined by ELISA. The IE promoter can direct the constitutive expression to yield about 0.4 g FK2P/10⁶ cells/day. The production of FK2P in these cell lines remains at about the same level after repeated passages, even in the absence of selective pressure. The FK2P accumulated in the culture medium is fully active in an assay using a chromogenic substrate for serine proteases. Western immunoblot analysis shows that the product remains predominately as single-chain molecules in serum-free medium, while in serum-containing medium two-chain material occurs as expected due to the presence of plasmin in serum. Judged from the size in Western immunoblots, the FK2P produced is glycosylated.     

A strategy for high cell density culture of heterotrophic microalgae with inhibitory substrates

Substrate inhibition is one of the major problems preventing high cell densities of microalgae in heterotrophic culture, so the possibility of overcoming the problem by various culture techniques was examined. It was found that perfusion culture may be the most appropriate technique for high cell densities in heterotrophic culture using inhibitory substrates. An experimental example in which a hollow fibre cell recycle system (HFCRS) was employed to achieve high cell densities of Chlamydomonas reinhardtii on acetate under heterotrophic conditions of growth was demonstrated. The cell density in the HFCRS was much higher than that reported in the literature for this species.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (q(O2) and q(CO2), respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate q(O2), q(CO2) and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), q(O2) and q(CO2) ranges were 5.14-5.77 and 5.31-6.36 pmol/cell day, respectively, resulting in RQ values of 0.98-1.14. Changes to DO had a slight reducing effect on respiration rates with q(O2) and q(CO2) values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with q(O2) and q(CO2) values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective q(O2) and q(CO2) values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in q(O2) and q(CO2) values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real-time q(O2) and q(CO2) estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing.     

The role of nerve cell density in the regulation of bud production in hydra

Summary The role of nerve cell density in the regulation of bud production in hydra was examined. Animals with different rates of bud production were produced by altering the temperature, population density and illumination of their cultures. When the distribution of cell types was examined in animals with different rates of bud production, the density of nerve cells in those animals was found to be correlated with their rate of bud production. Transfer of animals from one environment to another resulted in immediate changes in the rate of differentiation of large interstitial cells into nerve cells. This suggests that the density of nerve cells may play a role in regulating the rate of bud production in hydra.     

High cell density reactor for the production ofLactobacillus plantarum

Summary The production of a flocculent strain ofLactobacillus plantarum was performed in a high cell density reactor: a fluidized bed reactor (FBR) with a settler and an external cell recirculation. Two variables were assessed, the recirculation rate (R) and the dilution rate (D). The effect of the latter is much more important than the effect of the former in ensuring a quick start up in the flocculation process. The cell volumetric productivities obtained with this system increase directly with dilution rate and recirculation rate. The values of cell volumetric productivities obtained are considerably higher than those obtained in continuous stirred tank reactors (CSTR) and much higher than in batch reactors.     

A new culture system for the cultivation of mammalian cells for the production of several biologically active substances

A maximum cell density of 8×10⁶ viable human fibroblast cells/ml was obtained on microcarriers at a perfusion rate of 0.6 mL/min — a rate which maintained a quasi-steady state. The maximum tPA production was approximately 1.2 g/ml and obtained when the cell density was relatively constant during the later periods of cultivation. Specific UTP and tPA production rates had a correlation factor of 0.85, and cell growth to oxygen consumption had an 0.92 correlation factor. ATP generation was strongly correlated to glutamine consumption, with a lower correlation to oxygen utilization.     

In vitro culture of macrobrachium eggs

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infection by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the 3-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are irrigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979     

Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packed-bed culture system

Weakly adherent cells of the 293 line attached well to the internal surface of polyurethane foam (PUF) and grew to the high density of 6.83 × 10⁷ cells/cm³ PUF in stationary culture. The maximum productivity of tissue plasminogen activator (t-PA) was 0.158 IU/10⁶ cells per day. The productivity decreased at the stationary phase of cell growth, so we designed a PUF-plate packed-bed culture system for high density culture and continuous production of t-PA. A maximal cell density of 3.24 × 10⁷ cells/cm³ PUF and a t-PA productivity of 0.326 IU/10⁶cells per day were obtained in 25-day perfusion cultures. Although the cell density decreased to half that in PUF stationary culture, the t-PA productivity increased twofold and was maintained for 25 culture days.     

A simple and inexpensive multiple tube system for thein situ measurement of phytoplankton production by the14C method

A cheap and simple multiple tube incubation system is described for the in situ measurement of phytoplankton production by the ¹⁴C method. Incubation takes place in Pyrex glass tubes sealed with rubber suba-seal caps which are suitable for field, laboratory and incubator work. Comparison of production in the tubes with that in standard pyrex bottles showed no significant difference during both field and laboratory incubations. The in situ suspension system is transported in units and can be assembled into ladders in the field.     

Development of suspension culture of Podophyllum hexandrum for production of podophyllotoxin

A suspension culture of Podophyllum hexandrum was established. As the cultures grew, reduction in cell viability, biomass and product yield were associated with browning of culture medium, clumping of cells and drop in medium pH. Supplementation of the medium with both polyvinylpyrrollidone (PVP) and pectinase eliminated these problems. PVP at 10 g l^–1 was optimum for both growth of and product formation in P. hexandrum suspension cultures.     

In vitro culture of Montanoa tomentosa for the production of diterpenic acids

Following a solid phase extraction, GC-MS and GC-FID procedures, the production of three kaurane derivatives (grandiflorenic, kaurenoic and monoginoic acids) was detected in callus and cell suspension batch cultures of Montanoa tomentosa. From different hormonal combinations, the addition of 0.5 mg 2,4-dichlorophenoxyacetic acid l^–1 + 2 mg kinetin l^–1 increased the accumulation of total kaurenoids in 6 months old calluses to 2.1 mg g^–1 dry weight and in cell suspensions cultures up to 0.76 mg g^–1 dry weight. Monoginoic acid, which has not been detected before in leaves of wild plants, accumulated in both in vitro systems.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Evaluation of stable and highly productive gene amplified CHO cell line based on the location of amplified genes

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, variouskinds of stepwise methotrexate (MTX) selection were carriedout. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth andproduction rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage ofamplified genes near the telomeric region hardly changed, butthat of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productiveand stable gene-amplified cells based on the chromosomal location of the amplified gene.     

An oxygen supply strategy for the large-scale production of tissue plasminogen activator by microcarrier cell culture

An oxygen supply strategy involving agitation speed and aeration method for the large-scale production of tissue plasminogen activator (TPA) by a microcarrier cell culture was investigated by small-scale model experiments. A preliminary calculation indicated that diffusion limitation of dissolved oxygen (DO) could be caused in a microcarrier sedimentation layer more than 0.5 mm in thickness. Within an agitation speed range above 70 rpm, which was the critical speed for all of the microcarrier beads to remain suspended and thus for avoiding a deficiency of DO, the TPA productivity was higher at a lower agitation speed, while the cell concentration was not affected by the agitation speed. The addition of soluble starch to the culture medium prevented sedimentation of the microcarrier beads, even at the low agitation speed of 20 rpm, resulting in a TPA productivity higher than that at 70 rpm, which was the optimum speed without soluble starch. Use of an air spray system with an optimized air flow rate resulted in a k_La 2.35 times higher than that with simple surface aeration. Increasing the internal pressure of the culture from 0.2 kg/cm² (1209 hPa) to 1.5 kg/cm² (2483 hPa) had no effect on the cell growth but slightly increased the TPA production rates. However, based on the glucose consumption, both the cell and TPA yields were much improved by pressurization. As an optimum mixing and oxygen supply strategy for the production of TPA on a large scale, it is recommended that soluble starch be added to the culture medium to allow the microcarrier suspension to be maintained at a low agitation speed, while keeping a high oxygen transfer rate by means of an air spray system and pressurization.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.