Classifications

• • 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
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • 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
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes

Definitions

• the invention relates to a process for the separation of soluble cell contents from suspended cell debris or from suspended cells which release the soluble cell contents to the outside by selective adsorption of the cell contents on a solid support and subsequent detachment of the cell contents from the support.
• the object of the invention is to simplify, accelerate and efficiently design the known processes for separating soluble cell contents from suspended cell debris or from suspended cells which release soluble cell contents to the outside, and thereby to reduce the consumption of chro ⁇ reduce matographic material and solvents.
• the method according to the invention can be modified particularly simply and efficiently in that the Carrier has the features a) and b) and the carrier is separated from cells or cell debris by a combination of filtration on the one hand and draining or decanting on the other hand.
• all carriers which can sufficiently selectively adsorb the desired, dissolved cell contents can be used as carriers for the process according to the invention.
• a particular advantage of the method according to the invention is that it is also possible to work directly with supports which are suitable for affinity chromatography.
• soluble cell contents In order to obtain soluble cell contents from microorganisms, it is usually necessary to break up the cells and to separate the desired cell contents from this suspension of cell ruins. This method is used in particular in bacteria such as Escherichia coli if the soluble cell contents are peptides which are not released to the outside by this microorganism.
• the cells which release soluble cell contents, in particular peptides include, for example, yeasts.
• the soluble cell contents which can be separated off according to the invention include not only peptides but also other soluble cell substances with medium to higher molecular weights such as antibiotics, steroids, prostaglandins, antibodies, lectins etc. It is essential that these cell substances are soluble, and not only as a solvent Water and aqueous media, but also organic solvents come into question.
• SHEET In principle, all carriers which are suitable for the selective adsorption of soluble cell contents are suitable as solid carriers. If the cells or cell debris are to be separated only by filtration in accordance with case a), it only has to be ensured that the carriers have particle sizes larger by at least a factor of 5 than the cells or cell debris, and filter media are used which do indeed Allow cells or cell debris to pass through, but not the carrier. Carriers made of organic polymers, cross-linked dextrans, cellulose, cellulose derivatives, starch derivatives, etc. are also suitable for this.
• the carrier has a speci deviating by at least 0.2 g / l ⁇ fish weight than the cells or cell debris.
• Such deviations in the specific weight of the carrier can be achieved either by using fully or partially closed-cell foams or by carriers based on inorganic materials such as glasses, silicates, aluminates or polymerization with embedded, specifically heavy fillers, which also increase the specific weight of the particles accordingly .
• a material that has both the feature a) and the feature b) is, for example, glass, and this can be used in particular in the form of porous semolina.
• Semolina is offered commercially. If desired, its surface can be modified such that it only selectively adsorbs certain substances or groups of substances. Its surface can also be used for affinity chromatography by applying highly selective groups.
• Commercially available frits or sieves in particular glass frits, can be used as filter media, which admit the cells or cell debris but not the carrier. In order to avoid clogging of these glass frits by cells or cell debris, the pore size of the frits must be chosen to be sufficiently large. In order to avoid undesirable losses of carriers or clogging of the pores by the carrier, the particle size of the carriers must be at least 5 times larger
• a carrier is used which is 10 to 100 times larger than the cells or cell debris.
• the specific weight of the carrier must deviate sufficiently from the specific weight of the cells or cell debris.
• a deviation by the value of 0.2 g / l is sufficient, in particular if the carrier has particle sizes which are at least the same size, but preferably significantly larger than those of the cells or cell debris, since it is known that the speed of the flotation or sedimentation also depends on the particle size of the carrier. 5
• the flotation or sedimentation of the carrier can also be accelerated by centrifugation.
• a particular advantage of the method according to the invention is also that separation with centrifuges can be completely dispensed with and that otherwise Conventional two-stage separation in two separate apparatuses can be carried out according to the invention in one step and in one apparatus. While it has always been customary to separate the suspended cell debris or suspended cells before adding a solid support for adsorbing the cell contents, suspensions are produced according to the invention which contain both the cell debris or cells on the one hand and the solid support on the other, and then these to separate mixed suspensions in such a way that the solid supports are separated from the suspended cell debris or suspended cells. In terms of process engineering, such a separation is more difficult than the two successive separations of cell debris or cells from a suspension on the one hand and a solid support on the other.
• the method according to the invention leads to a number of unforeseeable advantages, so that it is entirely reasonable and economical to make use of the process measures which are unusual per se.
• the method according to the invention leads to lower losses and therefore higher yields of soluble cell contents.
• less solvent or washing medium is required according to the invention than according to the prior art.
• suspension can be repeated and separated again
• the working of invention when the carrier features a) and b), and therefore the separation of the carrier of the cells or cell debris by combining Fil ⁇ tration one hand and draining or decanting hand, ande can follow. It has proven particularly useful here to first make use of the flotation or sedimentation of the support and then to remove the remaining cells or cell debris by filtration. Both separation methods can be carried out in the same apparatus, which is also simple and inexpensive, since in principle it consists of only one vessel which has a frit bottom and a decanting or drain cock.
• soluble cell constituents are obtained from 5 suspended cells which release the soluble cell constituents to the outside, it is even possible to return the suspended cells to the fermenter and to use them again for production.
• a yeast cell suspension which was gen ⁇ by technological changes the yeast is able to synthesize a peptide and human ren to wirie- 5 was used.
• the approximately 40% yeast suspension was genograph homo ⁇ subsequent to the fermentation without any further intermediate step, and mixed with semolina in a stirred, cooled container, the peptide being bound to the semolina up to the saturation limit.
• yeast 30 as 0.5 g of yeast. It was then washed with 500 ml in the flow. 0.098 g of yeast were still found in the drain. 0.002 g of yeast was found on the semolina. The peptide was removed from the glass semolina with a suitable solvent eluted, a 3.75-fold enrichment compared to the suspension being found. The yield was about 10% higher than with a two-stage separation.
• Example 1 the glass semolina, as described in Example 1, was washed through a glass frit until the cell debris had been completely removed. In this combined process, the cell debris could be removed from the semolina with 30 to 50% less washing volume. The product was detached from the semolina as in Example 1.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Zoology (AREA)
• Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Wood Science & Technology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Mycology (AREA)
• Medicinal Chemistry (AREA)
• Biotechnology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Analytical Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=6361540

Family Applications (1)

Country Status (2)

Citations (1)

• 1988
  • 1988-08-25 DE DE19883828784 patent/DE3828784C1/de not_active Expired
• 1989
  • 1989-08-09 WO PCT/EP1989/000938 patent/WO1990002194A1/de not_active Ceased

Patent Citations (1)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events