WO2001005995A1 - Procede de preparation de surnageant de culture cellulaire - Google Patents

Procede de preparation de surnageant de culture cellulaire Download PDF

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Publication number
WO2001005995A1
WO2001005995A1 PCT/JP2000/004776 JP0004776W WO0105995A1 WO 2001005995 A1 WO2001005995 A1 WO 2001005995A1 JP 0004776 W JP0004776 W JP 0004776W WO 0105995 A1 WO0105995 A1 WO 0105995A1
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Prior art keywords
culture
cells
supernatant
phytase
yeast
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PCT/JP2000/004776
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English (en)
Japanese (ja)
Inventor
Kiyoshi Ito
Satoshi Yoshigai
Hitoshi Takahashi
Tadashi Araki
Junko Tokuda
Daisuke Mochizuki
Takeshi Nakamura
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Mitsui Chemicals, Inc.
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Priority to AU60186/00A priority Critical patent/AU6018600A/en
Publication of WO2001005995A1 publication Critical patent/WO2001005995A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • Ol igonucleotide to act as a PGR primer for amplifying of Phytase gene from Schwann iomyces occidental is
  • Oligonucleotide to act as a PCR primer for amplifying of Phytase gene from Schwann i omyces occidental is
  • the present invention relates to a method for producing a cell culture supernatant from which cells and nucleic acids have been removed.
  • Microorganisms are widely used as hosts for producing useful substances such as organic acids, amino acids, and proteins.
  • useful substances such as organic acids, amino acids, and proteins.
  • a technology for efficiently producing a useful substance using a transformed microorganism into which a gene for an industrially useful protein has been introduced by utilizing genetic engineering technology has become known. .
  • the microorganism When a microorganism is subjected to liquid culture and the microorganism secretes and produces a useful substance such as a protein outside the cell, when the microorganism is recovered (purified) from the culture, the microorganism must be removed from the culture. There is. In addition, depending on the purpose of use of the useful substance, it is important to remove foreign substances such as DNA fragments leaked from cells killed during the culture. In particular, when the intended useful substance is used for adding food or feed, and when a transformed microorganism is used, the transformed microorganism cells should be completely removed. Not only is essential, but also it is required not to mix the recombinant DNA fragment introduced into the transformed microorganism.
  • centrifugal separation, filtration, treatment with a flocculant, membrane separation, etc. are known as methods for removing microbial cells from a microorganism culture solution.
  • other methods than membrane separation completely remove the cells. Can not do it.
  • membrane separation microbial cells can be completely removed by using an ultrafiltration membrane such as the commonly used 1 ⁇ 1 "Membrane F" membrane, but DNA fragments
  • ultrafiltration membrane such as the commonly used 1 ⁇ 1 "Membrane F" membrane
  • microorganisms can be cultured at a high concentration (for example, 5 g ZL or more in dry cell weight) by fed-batch culture using glucose / methanol or the like as a carbon source, or can be cultured for a long period of one day or more (continuous culture).
  • a high concentration for example, 5 g ZL or more in dry cell weight
  • continuous culture there are cases where a large amount of viscous substances such as polysaccharides and high molecular components generated by the cells leaked from the cells killed during the culture and the cells are accumulated in the culture solution.
  • MF Two In order to almost completely separate and remove microbial cells from such cultures, MF Two
  • an object of the present invention is to remove not only cells but also nucleic acids such as DNA fragments when producing a culture supernatant from a cell culture of a microorganism or the like, and the filter is clogged.
  • An object of the present invention is to provide a method for producing a cell culture supernatant, which allows a target substance produced by a cell to be recovered at a high recovery rate without causing cell bleeding.
  • the inventors of the present application have found that by using a porous filter having a zeta potential, the recovery rate of the target useful substance is not reduced, and the filter is not clogged.
  • the present inventors have found that cells and nucleic acids can be removed, and have completed the present invention.
  • the present invention relates to a method for producing a culture supernatant from a cell culture, wherein the culture obtained by liquid culturing the cells or a processed product thereof is passed through a porous filter having a zeta potential.
  • a method for producing a culture supernatant which comprises the step of removing the cells and the nucleic acids.
  • the present inventors added a chitosan-based flocculant and a polyacrylic acid-based flocculant to a culture solution before subjecting the cell culture to one treatment with a porous filter having a zeta potential.
  • the cells and nucleic acids are firstly removed, and then the obtained primary supernatant is subjected to a single treatment with a porous filter having a zeta potential, without reducing the recovery rate of the target useful substance, and
  • the present inventors have found that cells and nucleic acids can be removed without clogging of filters, and the present invention has been completed.
  • the present invention relates to a method for producing a culture supernatant from a cell culture, wherein the culture obtained by liquid culture of the cells is 1) a chitosan-based culture medium or a processed product thereof.
  • a first step of adding a polyacrylic acid-based flocculant to aggregate the cells and nucleic acids 2) separating the aggregated cells and nucleic acids to prepare a primary supernatant, 3) The primary supernatant was used as a reagent having a zeta potential. Pass through L filter Three
  • the present invention provides a method for producing a culture supernatant, which comprises removing the cells and nucleic acids through the third step.
  • Cells in the cell culture used in the method of the present invention may be any of microbial cells, plant cells, and animal cells. Cells that produce the desired useful substance and secrete it into the culture supernatant are preferred, and in particular, microbial cells, particularly microbial cells that are transformed with a gene encoding the useful substance and produce the useful substance, are suitable. A simple example can be mentioned.
  • microorganisms examples include gram-negative bacteria such as Escherichia coli and Pseudomonas spp., Gram-philic fungi such as Bacillus sp. Lactic acid bacteria, yeasts such as Saccharomyces sp. And Actinomycetes such as Streptomyces sp.
  • Candida yeast is often used not only as a host for producing useful substances such as proteins and aldehydes, but also in recent years, utilizing genetic engineering technology, it has been possible to obtain genes for industrially useful proteins. Techniques for efficiently producing the useful substance using the introduced transformed Candida yeast have become known.
  • preferred microorganisms used in the present invention include Candida yeast, and Candida yeast transformed separately. Further, among the yeasts of the genus Candida, Candida boidini is a typical example.
  • the yeast of the genus Candida in the present invention is not particularly limited as long as it is capable of efficiently producing an industrially useful substance.
  • the gene of the above-mentioned useful protein is introduced by genetic recombination technology.
  • a preferred example is a transformed Candida yeast capable of efficiently producing the useful substance.
  • useful substances produced by cells are not limited at all, and examples thereof include enzyme proteins such as phytase, cellulase, xylanase, dalcanase, peroxidase, and oxidase, various amino acids, and various physiologically active peptides. Is not limited to these.
  • a cell culture or a processed product thereof is passed through a filter described below.
  • the cell culture means a culture in which the above-mentioned cells are cultured in a medium containing ordinary organic and Z or inorganic components.
  • the processed product of the cell culture means a product obtained by subjecting the cell culture to some treatment.
  • the processed product is subjected to centrifugation, filtration, coagulant treatment, etc.
  • Treatment of cell cultures from which cells have been primarily removed, cultures that have been sterilized in advance by chemical treatment or heat treatment, and cultures to which the above-mentioned useful protein substrates and inhibitors have been added in advance Means things.
  • a liquid containing a useful substance of interest immobilized in a carrier or in contact with cells cultured on the carrier is also included in the “cell culture or processed product thereof” of the present invention. Is done.
  • the nucleic acid in the present invention may be any nucleic acid contained in a cell culture or a processed product thereof, and may be any of DNA and RNA.
  • Typical examples are DNA fragments derived from chromosomal DNA molecules of cells, such as chromosomal DNA leaked out of cells and physically and chemically fragmented, and alkaline SDS treatment.
  • Examples include DNA molecules that are not insolubilized even after denaturation treatment of DNA molecules, DNA molecules that have not been insolubilized by a coagulant treatment, and DNA molecules that have not been supplemented by treatment with a resin such as a porous membrane or glass beads. .
  • examples thereof include a recombinant expression vector such as a recombinant plasmid vector or a recombinant virus vector used for the transformation and a fragment thereof.
  • a recombinant expression vector such as a recombinant plasmid vector or a recombinant virus vector used for the transformation and a fragment thereof.
  • nucleic acids having a size of 100 Kbp or less, more preferably 100 Kbp or less, and particularly about 100 to 10 Kbp can be removed.
  • r zeta filter a porous filter having a zeta potential
  • a porous filter having a zeta potential refers to not only a mechanical filtration action by a porous filter but also a culture or a cell and a nucleic acid in the processed product which are treated by the zeta potential of the filter. Means a porous filter that is also removed by being electrostatically adsorbed to the filter.
  • the zeta potential of the filter is not particularly limited, but is preferably +5 mV or more with respect to pure water having a pH of 7.0. Further, the pore size of the porous filter is preferably about 0.05 to 20 im.
  • Zeta filters usually have a basic structure of a porous matrix in which constituent fibers composed of cellulose, low melting point polyester, polyester, polyethylene, polypropylene or a combination thereof are mechanically entangled and Z or adhesively bonded between the fibers.
  • a modifier having a positive zeta potential in a neutral liquid is attached to the porous matrix.
  • the adhesive bonding between the fibers is performed by a method of adhesively bonding the fibers with an adhesive fiber, a method of adhesively bonding the fibers with an adhesive resin, or the like, but is not limited thereto.
  • Examples of the modifier for imparting a zeta potential include cationic colloidal silica and a polymer having a positive zeta potential in a neutral liquid.
  • preferred examples of the polymer include polyvinyl pyridine-styrene copolymer quaternary chloride, polycation-acrylic copolymer, polyallylamine, polyethylamine, polyethyleneamine, polyethyleneimine, polyamide-epiclorhydrin, and the like. It can be mentioned.
  • it is desirable that the water-soluble one is cross-linked with a cross-linking agent such as epoxy, melamine, aldehyde, or the like, and is insoluble in water. As described above, it is preferable that these modifiers can impart a zeta potential of +5 mV or more to neutral water.
  • the modifier having a positive zeta potential can be attached relatively uniformly to the surface of the porous matrix.
  • the zeta potential can be measured by an ordinary method, and a device for that is also commercially available (for example, an ultrasonic zeta potential measuring device (model: Acoustophor 8000) manufactured by Penkem, a laser zeta manufactured by Coulter) Potential measuring device (model: DELSA-440), laser zeta potentiometer manufactured by Otsuka Electronics Co., Ltd. (model: ELS-8000, ELS-6000, etc.) Zeta potential can be easily measured using such a commercially available device. .
  • an ultrasonic zeta potential measuring device model: Acoustophor 8000 manufactured by Penkem
  • Potential measuring device model: DELSA-440
  • laser zeta potentiometer manufactured by Otsuka Electronics Co., Ltd. model: ELS-8000, ELS-6000, etc.
  • Zeta potential can be easily measured using such a commercially available device. .
  • the principle of measuring the zeta potential by a conventional method is as follows. In other words, when two phases are in contact with each other, when a voltage is applied externally between them, both phases move relative to each other, or conversely, both phases move relatively.
  • the electrokinetic phenomenon in which an electric field appears in the direction parallel to the interface, was measured, and the zeta potential, which indicates the charge state at both interfaces, was indirectly determined from each calculation formula using the measurement results. For example, when the streaming potential is used, the zeta potential is obtained by the following equation (1).
  • ⁇ ⁇ ( ⁇ ' ⁇ ) / ( ⁇ 0 ⁇ ⁇ ⁇ ) ⁇ - ⁇ / ⁇ ⁇ ⁇ (1)
  • the conductivity ⁇ is based on Ohm's law used in the process of deriving the equation, and is proportional to the magnitude of the streaming potential generated by the movement of the electric charge at the interface electric double layer. Used as a count.
  • the dielectric constant ⁇ and the conductivity ⁇ must accurately consider the dielectric constant and conductivity of the interfacial electric double layer.However, assuming that they are equal to the dielectric constant and conductivity of the liquid, The values described in the literature for pure liquids are used.
  • the zeta potential has been measured and calculated as described above.
  • ⁇ ⁇ ⁇ ⁇ in equation (1) is measured with a pair of electrodes installed in the measurement system. Since this E depends on the dielectric constant and the electrical conductivity between the electrodes in principle, the value of E slightly varies depending on the measurement system. Since phenomena in the system interact, strictly speaking, the values of the dielectric constant and conductivity should assume both the electric charge of a liquid and a solid, but in general, a pure liquid
  • the zeta potential is determined using the literature value of.
  • the pore size of the L-type matrix may be evenly distributed, but should the pore size on the inflow side be larger than on the water outflow side? It is desirable that the L diameter is distributed. With such a structure, relatively large particles that cause contamination are collected on the inflow side, and relatively small particles are collected on the outflow side, so that clogging hardly occurs. Or many with multiple zeta potentials with different average pore sizes? The same can be achieved with an L filter.
  • the first porous filter having a zeta potential having a large average diameter for example, about 5 to 2 Om in average diameter
  • a first porous filter having a smaller average diameter for example, By treating with a second porous filter having a zeta potential, clogging hardly occurs and the life of the filter can be extended.
  • a plurality of filters are used, they can be used by arranging them in series in descending order of average pore diameter from the upstream side.
  • the zeta filter itself is well known and is commercially available.
  • a commercially available porous filter that claims to cause adsorption based on zeta potential satisfies the above-mentioned zeta potential of +7.0 mV with respect to pure water having a pH of 7.0, and the method of the present invention.
  • An example of one such commercially available filter is the Zetaplus (registered trademark) series, manufactured by CUNO, IC. Of Connecticut, USA (and Cuno Corporation of Yokohama, Japan, a Japanese corporation), ie, Zetablass® filters S Series, C Series, A, Series, U Series, LA Series and the like. Since these have a lineup of those having various pore sizes, desired filters can be used in combination as appropriate as described above. 8
  • a preferred example is a method of adding a chitosan-based and / or Z- or polyacrylic-acid-based flocculant to the culture solution or the processed product of the culture solution.
  • Coagulants are used in industrial wastewater such as papermaking, mining, civil engineering, cement manufacturing, aluminum refining and food processing, as well as in the sedimentation and dehydration of inorganic and organic sludge in the process. Widely used for etc. In other words, if the size of the solid suspended in the liquid is about 1 or less, it becomes difficult to separate the solid by physical methods such as ordinary precipitation and filtration. Have been. Chemicals that aggregate suspended fine particles into large aggregates, thereby facilitating sedimentation and filtration are called flocculants. Organic and inorganic flocculants are used.
  • a chitosan-based flocculant and a Z or acrylic acid-based flocculant, which are organic polymer-based flocculants, are used.
  • Chitosan is obtained by heating chitins of crustaceans such as renji and shrimp with a concentrated alkaline solution to at least partially deacetylate the acetylamino group attached to the cyclic portion of the sugar unit of chitin. Things.
  • the acetylation rate of the chitosan used is not particularly limited, but is preferably about 67 to 85 mol%.
  • such chitosan can be preferably used as a flocculant.
  • a chitosan derivative in which an anionic group, for example, a carboxyl group, a sulfone group, or the like is introduced into chitosan can be preferably used.
  • Chitosa 9 Chitosa 9
  • the chitin-based flocculants include chitosan itself and its derivatives.
  • the chitosan used as the coagulant preferably has a large molecular weight, more preferably 20,000 or more, particularly preferably about 50,000 to 400,000.
  • a chitosan-based flocculant itself preferably used in the method of the present invention is well known, is commercially available, and a commercially available product can be preferably used.
  • a commercially available product there may be mentioned, for example, Crime Bar 101 manufactured by Kurita Kogyo Co., Ltd. of Tokyo, Japan, but is not limited to this.
  • polyacrylic acid-based flocculant a polymer comprising a repeating unit represented by the following general formula can be preferably used.
  • X is a substituent selected C00H, G00Na, from C00NH 2.
  • X is polyacrylic acid (anionic For G00H), polyacrylic acid sodium For GOONa (cationic) And polyacrylamide (non-ionic), each of which is used as a polyacrylic acid-based flocculant
  • the molecular weight of the polyacrylic acid-based flocculant is not particularly limited, but is preferably as large as possible. Usually, about 200,000 to 100,000 is preferable.
  • Such a polyacrylic acid-based flocculant itself preferably used in the method of the present invention is well known, commercially available, and a commercially available product can be preferably used.
  • a commercially available product Crimbar 201 manufactured by Kurita Kogyo Co., Ltd. of Tokyo, Japan can be exemplified, but of course, the invention is not limited to this.
  • any of a chitosan-based flocculant alone, a polyacrylic acid-based flocculant alone, or a combination of two kinds of chitosan-based and polyacrylic acid-based flocculants may be used.
  • the amount of the chitosan-based flocculant used is preferably 0.1 to 8% by weight, more preferably 1 to 6% by weight, based on the amount of cells contained in the culture solution (in terms of dry weight). % By weight.
  • the amount of the polyacrylic acid-based flocculant used is preferably 0.05 to 2.0% by weight / o with respect to the amount of cells contained in the culture solution.
  • the p H of the culture solution sulfuric acid, propionic acid, formic acid, from p H 2.
  • Pre-adjustment under acidic conditions of 5 to 5.0 improves the cell aggregation effect.
  • the order of addition is not particularly limited. However, when adding these, it is easier to agglutinate the cells by stirring as strongly as possible.
  • the liquid thus treated with the flocculant is separated from the cell culture by centrifugation, filtration, decantation, etc., and is not separated from the primary supernatant by the zeta filter treatment described above. Cells and nucleic acids may be removed.
  • the cells in the cell culture to be subjected to the method of the present invention are not particularly limited as described above.
  • a transformed candy producing a useful substance as described above is preferable.
  • Da yeast can be mentioned.
  • As an example of such transformed Candida yeast Candida boidini i MT-40544 strain (FERM BP-5936) (EPM) into which a phytase gene derived from Schwanniomyces occidental is introduced has been introduced. 0 931 837 A1), but of course, the present invention is not limited to this, and the same form can be applied to microorganisms in general.
  • the composition of the liquid medium for culturing Candida yeast is not particularly limited as long as Candida yeast grows smoothly and useful substances are produced in the culture medium.
  • the carbon source include methanol, glycerol, ethanol, glucose and the like.
  • the nitrogen source various organic or inorganic nitrogen sources such as peptone, corn steep liquor, yeast extract and ammonium sulfate can be used alone or in combination. Also, salts of metals such as magnesium, sodium and potassium (phosphate, sulfate, hydrochloride), trace amounts of gold 1 1
  • a variety of vitamins, nucleic acid-related substances, pH regulators, etc. can be added to the medium.
  • the liquid culture method is 15 to 35 ° C, preferably 25 to 30 ° C.
  • the pH of the culture solution during the liquid culture is not particularly limited as long as the pH can grow the present yeast, but is usually in the range of pH 2 to 8, more preferably pH 4 to 7.
  • the culture period is usually about 1 day to 60 days, preferably about 7 days to 40 days. When the yeast cells and the supernatant are separated from the liquid in the continuous culture, the culture period is not particularly limited.
  • Candida boidinii MT-40544 (FERM BP-5936) was inoculated and cultured at 27 ° C for 2 days with shaking.
  • the culture was subjected to primary filtration using a Zetaplus filter BioCap 01A (particle size: 5 to 20 m, 24.6 cm 2 ) manufactured by Cuno Co., Ltd., which was washed with sterile water.
  • the obtained filtrate was filtered through a zeta plus filter BioCap10S (particle diameter: 0.9 to 2 im).
  • the filtrate obtained here is used as a Zetaplus filter BioCap30S (particle diameter 0.6 to 1.5 m), and the same BioCap60S (particle diameter 0.2 to 0.5). ⁇ m).
  • the number of viable yeast in 0.1 ml of the filtrate at each filtration stage was confirmed using a solid medium containing 1.5 ⁇ 1 ⁇ 2 agar in a culture solution having the composition shown in Table 1, and Separately, the amount of phytase protein in the filtrate was measured. Table 3 shows the results. At this time, the recovery rate of the phytase protein by the final 4-stage treatment of the zeta filter was 91%. 13
  • the culture termination solution obtained in Example 1 was centrifuged (9000 X g, 5 minutes) to remove yeast cells primarily. This centrifuged supernatant was divided into four tubes of 20 OmI each. Each of the supernatants was washed with sterile water, and the ZetaPlus filter BioCap (24.6 cm2) manufactured by Cuno Co., Ltd. was used for 01A (particle size 5 to 2 Oyum) and BIOcap 30S (particle size). 0.6 to 1.5 / im), BioC ap 60 S (particle size 0.2 to 0.55 m), BioC ap 90 S (particle size 0.05 to 0.2 m) ).
  • the number of viable yeast in 0.1 ml of each filtrate was confirmed using an individual medium obtained by adding 1.5% agar to the liquid medium shown in Table 1, and the amount of phytase protein in the filtrate was measured at the same time. .
  • Table 4 shows the results. No live yeast was detected in the 60 S filtrate, and the recovery of phytase protein at this time was 93%. Even when using any filter, supply pressure filtration end plate remained within 0. 5 k gZcm 2. 14
  • Oligonucleotides having a base sequence were used.
  • the Perkin-Elmer Corporation 3 ⁇ 4 (0.5 U CD Amp Ii Taq Gold.
  • the PCR reaction was performed under the condition that a reaction cycle consisting of 72 ° C. and 1 minute was repeated 25 times. (Galose concentration 0.8%), the presence of the amplified fragment could not be confirmed.
  • Example 2 The same centrifugal supernatant 2 OOm I as used in Example 2 was converted to a microza pencil type module PS of Asahi Kasei Kogyo Co., Ltd., which is a cruz flow type ultrafiltration membrane without zeta potential. Filtration was performed with P-013 (MF, 80 cm 2 , particle size 0.1 l / ⁇ m). Since the back pressure at the end of filtration became 1 kgZcm 2 or more, which is the maximum working pressure, the filtration operation was stopped when the liquid volume at the start was approximately 6 times concentrated.
  • P-013 MF, 80 cm 2 , particle size 0.1 l / ⁇ m
  • the number of viable yeast in 0.1 ml of the filtrate was confirmed using a solid medium containing 1.5% agar in a liquid medium having the composition shown in Table 1. No live yeast was detected. When the amount of phytase protein in the filtrate was measured, the recovery of phytase protein was 45%. Table 5 shows the results. Table 5
  • a chitosan diluent was prepared by mixing 98.5 g of pure water with 0.5 g of Crime Bar 101 manufactured by Kurita Water Industries Ltd. and 1 g of acetic acid.
  • the amount of the above-mentioned diluted chitosan solution was changed to 30 g , 45 g, and 60 g, and the mixture was stirred vigorously for 5 minutes.
  • This treated solution was centrifuged to remove bacteria (about 3000 X g, 10 minutes), and the turbidity (absorbance: 660 nm) of the supernatant was measured to confirm the cohesiveness of the yeast cells.
  • the addition of the chitosan-based flocculant reduced the turbidity of the supernatant, and confirmed the aggregation of yeast cells.
  • the chitosan diluent was prepared by adding 98 g of pure water to Crime Bar 101 (made by Kurita Water Industries Ltd.) 1 7
  • the polyacrylic acid diluent was prepared by dissolving 0.2 g of Crime Bar 201 (manufactured by Kurita Water Industries Ltd.) in 99.8 g of pure water. Propionic acid was used as a pH adjuster.
  • 0.7 g of the chitosan diluent was added to each 1 Og of the culture termination solution obtained by culturing in the same manner as in Example 1 when the pH was not adjusted and when the pH was adjusted to 4.2.
  • the mixture was vigorously stirred for 5 minutes, and then 0.7 g of a diluted polyacrylic acid solution was added.
  • the mixture was stirred until uniform formation of floc and floc was confirmed, and allowed to stand for 30 minutes.
  • This treated solution was subjected to centrifugal eradication treatment (about 2000 X g , 10 minutes), and the turbidity (absorbance: 660 nm) of the supernatant was measured to confirm the cohesiveness of the yeast cells.
  • Table 8 when the pH was adjusted to 4.2, the turbidity of the supernatant liquid was reduced, and it was confirmed that the yeast cells were better aggregated.

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Abstract

L'invention concerne un procédé de préparation d'un surnageant de culture cellulaire exempt de cellules et d'acide nucléique, sans encrassement du filtre. Ledit procédé permet l'obtention d'un pourcentage de récupération élevé d'une substance utile voulue produite par les cellules. Dans ledit procédé, les cellules et l'acide nucléique sont enlevées par le passage d'un produit de culture ou son produit traité obtenu par culture en liquide, dans un filtre poreux à potentiel zêta.
PCT/JP2000/004776 1999-07-15 2000-07-14 Procede de preparation de surnageant de culture cellulaire WO2001005995A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020000149A (ja) * 2018-06-29 2020-01-09 ナガセケムテックス株式会社 細胞生産物の製造方法
CN115669542A (zh) * 2022-11-04 2023-02-03 西北农林科技大学 一种植物组织培养脱毒方法

Citations (4)

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Publication number Priority date Publication date Assignee Title
JPH03201977A (ja) * 1989-12-28 1991-09-03 Kobayashi Pharmaceut Co Ltd 血中微生物培養方法及びその方法に用いる培養器
JPH04197410A (ja) * 1990-11-28 1992-07-17 Unitika Res Lab:Kk 液体用多層構造濾過布
JPH08289782A (ja) * 1994-07-05 1996-11-05 Mitsui Toatsu Chem Inc 新規なるフィターゼ
JPH11206368A (ja) * 1998-01-28 1999-08-03 Mitsui Chem Inc フィターゼの分泌生産方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03201977A (ja) * 1989-12-28 1991-09-03 Kobayashi Pharmaceut Co Ltd 血中微生物培養方法及びその方法に用いる培養器
JPH04197410A (ja) * 1990-11-28 1992-07-17 Unitika Res Lab:Kk 液体用多層構造濾過布
JPH08289782A (ja) * 1994-07-05 1996-11-05 Mitsui Toatsu Chem Inc 新規なるフィターゼ
JPH11206368A (ja) * 1998-01-28 1999-08-03 Mitsui Chem Inc フィターゼの分泌生産方法

Cited By (4)

* Cited by examiner, † Cited by third party
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JP2020000149A (ja) * 2018-06-29 2020-01-09 ナガセケムテックス株式会社 細胞生産物の製造方法
JP7132771B2 (ja) 2018-06-29 2022-09-07 ナガセケムテックス株式会社 細胞生産物の製造方法
CN115669542A (zh) * 2022-11-04 2023-02-03 西北农林科技大学 一种植物组织培养脱毒方法
CN115669542B (zh) * 2022-11-04 2023-08-25 西北农林科技大学 一种植物组织培养脱毒方法

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