DE10329819B4 - Process for the extractive isolation of organic components from cell suspensions and cell-free media - Google Patents

Abstract

method for the isolation of by biocatalytic conversion by means of Yeast cells produced xenobiotic organochemical compounds by extraction with an organic solvent from cell suspensions or after a cell separation step from cell culture media, thereby characterized in that prior to extraction of the cell suspension or the cell culture medium for enzymatic degradation of the yeast cells excreted bioemulsifiers a hydrolase of at least a protease and / or glycosylase is added and after a subsequent incubation step the extraction with the organic solvent takes place.

Description

The The invention relates to a process for the extractive isolation of organic chemical Components produced by microorganisms (biotransformation) are from cell suspensions and cell-free media. This method is especially interesting for the microbial production of chiral intermediates for the pharmaceutical Industry.

US Pat. No. 2,471,597 discloses a method for liquid extraction of penicillin from an aqueous culture fluid. The natural product penicillin is obtained by fermentation of the penicillin-producing molds Penicillium notatum and Penicillium chrysogenum.

GB 1 238 836 describes an extraction process for the natural product erythromycin from a Streptomyces erythreus culture broth with organic solvents.

In GB 1 288 843 describes a method which improves the yield of fermentation products, in particular antibiotics such as tetracycline, streptomycin, oleandomycin and penicillin.

In GB 1 230 065 discloses a method by which double-stranded RNA can be isolated by extraction with the organic solvent phenol from a fermentation broth of E. coli cells.

Derwent Abstract No. 1987 - 32 5809/46 describes the purification of by fermentation of Xanthomonas Campestris produced polysaccharides, especially xanthan.

At the in US Pat. No. 2,471,597 . GB 1 238 836 . GB 1 288 843 . GB 1 230 065 and Derwent Abstract No. 1987-325809 / 46 are processes for the extractive isolation of fermentation products, but not for methods for the extractive isolation of biotransformation products.

The biotechnological-chemical synthesis of chemicals with the help of Microorganisms are known. This is a biocatalytic Substitution (biotransformation) of the starting chemicals in the Cell by cellular enzymes. Main application of this technology is the production of chiral pharmaceutical intermediates (CPI). Downstream processes for This technology is known from the literature and also as a result of its own Works published (Haberland et al, 2002, Bertau, 2001, Davoli et al., 1999 and references cited therein).

typically, the organic components become after a cell separation step with organic solvents extracted from the cell-free medium. This usually requires an up to tenfold surplus on organic solvent to the aqueous Phase can be added.

Problematic is that the addition of the organic solvent causes gel and slime formation phenomena. These represent a key complication in isolating the microbially produced substances from whole-cell biotransformations and sometimes have drastic effects on the recovery rate.

To The prior art is in the reductive biotransformation of 2-cyclopentanonecarboxylic acid ethyl ester the product (1R, 2S) -cis-2-hydroxycyclopentanecarboxylic acid ethyl ester typically obtained in 64% yield.

The Emulsification problem is usually solved by centrifugation of the ether / water mixtures (Davoli et al., 1999 and references cited therein). While this method is already in the laboratory scale is clearly dangerous, it is even less suitable on a large scale.

alternative are the crude product-containing culture broths, optionally after one Cell separation step, concentrated to dryness and then with an organic solvent extracted. The product becomes lipophilic cell components to a considerable extent contaminated, which makes expensive cleaning operations necessary.

The prior art usually employs a thermal cleaning process (distillation) to separate the desired product from the lipophilic cell constituents. For steam-volatile Compounds are observed in this method, depending on their steam volatility partial loss rates of up to 90%. In order to separate the nonvolatile product by distillation from the lipophilic cell constituents high temperatures (70 ° C to 130 ° C) are necessary regularly even in a high vacuum.

moreover can Thermal by-products produce reactive by-products resulting in lower maximum product purities, partial Loss of stereoisomeric purity and up to 80% loss desired Product.

The Products of whole-cell biotransformations are becoming more prevalent Majority for the production of pharmacological agents used, resulting in Episode has this Problems in product recovery are not just economic, but also on the pharmacological activity of the final product impact. In addition, the formation of reactive species poses a risk potential in the field of occupational safety.

Of the Invention is based on the object, an improved method for the isolation of organic components from microbiological Indicate yeast cell suspensions and cell-free culture media in particular chemical modifications to the functional groups of the desired Products are avoided. About that In addition to the invention, a continuous or semi-continuous Extraction from ongoing cultures may be possible.

According to the invention Task solved by a method of isolating by biocatalytic material conversion xenobiotic organochemical produced by yeast cells Compounds by extraction with an organic solvent from cell suspensions or after a cell separation step Cell culture media, wherein prior to extraction of the cell suspension or the cell culture medium for enzymatic degradation of the yeast cells excreted bioemulsifiers a hydrolase of at least a protease and / or glycosylase is added and after a subsequent Incubation step extraction with the organic solvent he follows.

Surprisingly could as cause for the gel and slime formation bioemulsifiers of biotransformation used microorganism can be identified.

Amazingly, can by the treatment of the cell-free culture medium preceding the extraction step or the cell suspension with a hydrolase component, the bioemulsifiers almost complete be removed from the cell culture medium or the cell suspension.

This is surprising since the bioemulsifiers present in the microorganisms are not considered to be natural Substrates for Hydrolases are known. In addition, it was feared that the bio-emulsifiers by their emulsifying effect a danger to the structural integrity of the hydrolases represent.

Advantageous remains by the inventive enzymatic Degradation of Bioemulgatoren observed in the prior art Problem of gel and slime formation on addition of the extractant. In the method according to the invention let yourself during extraction no significant conversion of lipophilic cell components prove in the organic phase.

simultaneously is in the process of the invention with a quantity of solvent, which is smaller than the watery one Phase observed a significant phase separation. From ecological and economic reasons in the process according to the invention volumes of organic solvent used are therefore preferred smaller than the volume of cell culture medium used for extraction or the cell suspension.

thanks in the inventive method Lack of gel and slime formation contains the organic phase after extraction of almost pure product in maximum yield. Thereby can even go to a purification step, depending on the state the technique of extraction adjoins. To extraction has to be done only for extraction volatile solvent in usual Way, z. B. by distillation, from the low volatility Product component are separated. This separation can be gentle at low temperatures (25 ° C up to 35 ° C) and pressing (600-1030 hPa).

By the saving of costly cleaning steps will and reduces material costs and increases the yield of product. simultaneously also reduces due to the lack of gel and slime formation the amount of solvent necessary for the extraction. Is advantageous the solvent used for extraction after separation of the product in the process according to the invention so pure that it for Subsequent extractions are used again without prior cleaning can or circulated can be. The resulting reduction in spent solvent is another economic and more ecological Advantage.

Thereby, that with the inventive method to a thermal purification step (distillation) for purification can be dispensed with the product occur in the process of the invention no thermally induced side reactions. Becomes advantageous thereby increasing the yield and purity of the product and in particular the formation of unwanted Prevents stereoisomers.

By the waiver of the thermal cleaning step is also the Occupational safety significantly increased with the method according to the invention, there now Also thermolabile products without the risk of formation reactive By-products can be worked up.

In a particularly advantageous embodiment of the invention takes place the extraction directly from the cell suspension without a previous one Cell separation step.

Thus, as illustrated by the following flow scheme, the method steps which are necessary for the isolation of organic components from the culture medium or the cell suspension are considerably reduced by the method according to the invention. In the left column, the steps of the method according to the invention are listed; in the right column, the isolation steps, which are usually carried out according to the prior art:

Prefers the yeast cells of the genus Saccharomyces cerevisiae belong.

Under Hydrolases are enzymes that convert the substrates into a reversible one Hydrolytic cleavage reaction (hydrolysis), such as. Lipases, glycosylases or proteases.

The Selection of the method according to the invention used enzymes is by the microorganism and the to be isolated Product determined. The hydrolase component consists of one or several enzymes.

in the inventive method is a hydrolase of at least one protease and / or Added glycosylase.

It is known that most hydrolases in addition to their actual enzymatic specificity also hydrolyze ester groups. This is problematic because the microbially produced organochemical Ver bonds often hydrolysis-sensitive groups, such as. As ester groups, whose hydrolysis must be avoided.

Surprised It was found that the hydrolysis of the organic chemical Compounds contained ester groups in the process according to the invention completely can be avoided.

So can be found in the use of glycosylases and proteases from the group the serine proteases or metalloproteases in the process according to the invention the degree of hydrolysis of ester groups in the product to be isolated kept zero.

This is surprising before the background determined in own experiments that at the use of cysteine proteases, such as. B. Papain, a significant Hydrolysis of the ester groups in the product to be isolated from up to 30% was observed.

in the Case that the product to be isolated hydrolysis sensitive groups contains the hydrolase therefore preferably consists of glycosylases and / or Proteases from the group of serine proteases or metalloproteases.

Prefers used proteases from the group of serine proteases are: α-chymotrypsin, kallikrein, Pronase E and subtilisin.

Prefers used proteases from the group of metalloproteases are: carboxypeptidase and thermolysin.

preferred Glycosylases are β-galactosidases and pectinases.

The Conditions of incubation with the hydrolase component may be advantageous adapted to the microorganism and isolating product.

Preferably the incubation happens at conditions that are the usual Culture conditions of the microorganisms used for biotransformation correspond. This can be advantageous incubation with the Hydrolasekomponente performed parallel to the cell culture become. For this purpose, preferably the hydrolysis component is located while the entire biotransformation in catalytically sufficient amount in the cell culture medium.

Prefers is the incubation at a temperature of 25 ° C to 45 ° C, and a pH between pH 5 and pH 8 performed. The medium is the Hydrolasekomponente with an enzyme activity of preferably 10 units to 100 units per ml added.

in the Case of post-biotransformation incubation with the hydrolase component is preferably carried out in a period of time up to 5 hours.

Surprised it was found that the presence of the hydrolase in the cell culture medium also about one long period, from up to several days, do not interfere cell culture and biotransformation. Amazingly, Hydrolase even has a positive effect on certain substrates Effect on the biotransformation itself.

So was surprisingly found in the microbial conversion of keto substrates with dehydrogenases, that upon addition of the bio-emulsifier-cleaving hydrolase at the beginning of the Cell culture bioavailability the substrates can be increased.

presumably This is due to the fact that by the hydrolase-related enzymatic degradation of the bioemulsifiers the otherwise observed bioemulsifier - mediated hydration of the Keto substrates to geminal diols is significantly minimized. The Geminal diols, in contrast to the keto substrates, are not substrates for dehydrogenases.

In a particularly preferred variant of the method is therefore the Hydrolase already at the beginning of the biotransformation or the cell culture added to the medium.

Advantageously, the presence of the hydrolase during the biotransformation of Ke to substrates higher conversion rates or shorter reaction times realized.

The Product is also in greater purity and stereoisomeric purity - with all the benefits for the pharmaceutical Final product.

In a particularly advantageous variant of the method according to the invention is during cell culture, the cell suspension or the cell culture medium continuously incubated with the hydrolase component. For this purpose, the hydrolase is the same time added with the starting material of the biotransformation of the cell culture. Parallel For biotransformation, the product of biotransformation becomes this variant is continuous or semi-continuous from the Cell suspension or the cell culture medium isolated by extraction.

These continuous incubation and extraction is thereby made possible that, as previously explained, the Incubation with the hydrolase more surprising There is no negative effect on cell culture and biotransformation shows.

By subsequent embodiments the invention will be closer explains:

Embodiment 1: Biotransformation

For the reduction of 2-cyclopentanonecarboxylic acid ethyl ester to (1R, 2S) -cis-2-hydroxycyclopentanecarboxylic acid ethyl ester in a Ganzzellbiotransformation with Saccharomyces cerevisiae procedure is as follows:
200 g of baker's yeast (Saccharomyces cerevisiae) from Fala, F-Strasbourg and 200 g of sucrose are introduced into a fermenter filled with 10 l of tap water. After 20 min ,. Incubation at 30.0 ° C is added to the aerated culture (2.0 l of air per liter of solvent and minute) of 100 ml of 2-cyclopentanonecarboxylic acid ethyl ester (equivalent to 107.9 g or 691 mmol) continuously over 20 h, the pH at 6.3 was held. The reaction is monitored by gas chromatography.

Embodiment 2: Product Isolation from cell suspension

For product isolation from the culture broth obtained according to Example 1, the cell suspension after 20 h of biotransformation carried out according to Example 1 for biocatalytic Biodeemulgation with the protease kallikrein with an enzyme activity 60 kU / l, incubated for 5 h at 30 ° C and after complete phase separation (38 h) extracted with methyl tert-butyl ether (MTBE). After drying over MgSO ₄ , the volatiles are removed in vacuo.

• Aspekt Farblose Flüssigkeit mit charakteristischem Geruch
• IR-Spektrum entspricht Referenzspektrum
• Gehalt (Summe der Isomeren) (GC) ≥ 97 %
• Enantiomerenüberschuss (ee) ≥ 98 %
• Diastereomerenüberschuss (de) ≥ 94 %
• Siedepunkt 70 °C–72 °C (4 mm Hg)
• Brechungsindex
  
  1.456
• Drehwert [α]
  
  15° ± 2°
• Drehwert [α]
  
  18° ± 2°

The product, (1R, 2S) -cis-2-hydroxycyclopentanecarboxylic acid ethyl ester, is obtained in this 89% yield and meets the following specifications:

• Aspect Colorless liquid with a characteristic odor
• IR spectrum corresponds to reference spectrum
• Content (sum of isomers) (GC) ≥ 97%
• Enantiomeric excess (ee) ≥ 98%
• Diastereomeric excess (de) ≥ 94%
• Boiling point 70 ° C-72 ° C (4 mm Hg)
• refractive index
  
  1456
• Rotation value [α]
  
  15 ° ± 2 °
• Rotation value [α]
  
  18 ° ± 2 °

The enantiomeric excess (ee) is defined as follows: ee = ((1R, 2S) - (1S, 2R)) / ((1R, 2S) + (1S, 2R)) in%

The diastereomeric excess (de) is defined as follows: de = ((1R, 2S) - (1S, 2S) - (1R, 2R)) / ((1R, 2S) + (1S, 2S) + (1R, 2R)) in%

It can be seen from the data that the desired isomer (1R, 2S) -cis-2-hydroxycyclopentanecarboxylic acid ethyl ester in almost stereoisomeric purity (≥ 94%) is obtained by the process according to the invention is.

Embodiment 3: Influence on phase separation

The Influence of the process of the invention for product isolation with biocatalytic deemulsification was used on Example of 2-cyclopentanonecarboxylic acid ethyl ester on the phase separation in the extraction for various hydrolases determined.

The Presence of emulsifiers in the extraction lowers the efficiency the extraction in terms of quantity and purity of the product to be isolated. At the same time leads the presence of emulsifiers to the formation of over several weeks or months of stable gels or mucus.

The Phase separation time can therefore be used as a measure of the presence of emulsifiers and the efficiency of the extraction can be used.

To was a biotransformation, as described in Example 1 carried out. The according to embodiment 1 culture broth obtained was used for product isolation analogous to Example 2 for the biocatalytic De-emulsification with a hydrolase added. Here, however, were the Type and concentration of hydrolase used for biodegradation varies and isolation from cell suspension and cell culture medium compared:

3a) Isolation from cell culture medium:

• ¹ t_i – Inkubationszeit
• ² t_p – Phasenseparationszeit
• ³ ohne Enzymzugabe

For product isolation from cell culture medium, the cell suspension obtained according to Example 1 was centrifuged at 10,000 g. Immediately after completion of the centrifugation, the respective enzyme indicated in Table 1 was added to 5 ml each of the cell culture medium in the centrifugation supernatant. Thereafter, the medium was incubated at pH 6.6 and 30 ° C for the incubation time (t _i ) given in Table 1. Subsequently, the incubated cell culture medium was overlaid with 2 ml of methyl tert-butyl ether (MTBE), and the phases with the vortexer (40 Hz) for 30 sec. Mixed. Subsequently, the phase separation time is optically determined by the occurrence of a clearly recognizable phase boundary. Table 1: Results of the hydrolase-catalyzed cleavage of the bioemulsifier of Saccharomyces cerevisiae for cell-free medium (pH 6.6, 30 ° C).

• ¹ t _i - incubation
• ² t _p - phase separation time
• ³ without enzyme addition

The Results summarized in the table clearly show that the phase separation time by incubation with hydrolases clearly shortened can be. This is evidence of the deemulsive influence the hydrolases and shows that with the use of hydrolases the Efficiency of extraction regarding quantity and cleanliness of the product to be isolated significantly increased can.

3b) Isolation from cell suspension:

The Isolation was carried out analogously to 3a. described, but without prior Separation of biomass around the influence of cells and cell wall components to determine the bioemulsification.

• ¹ t_i – Inkubationszeit
• ² t_p – Phasenseparationszeit
• ³ ohne Enzymzugabe

In contrast to 3a, the centrifugation step was omitted and the enzyme concentration given in Table 2 was added directly to in each case 5 ml of cell suspension. Table 2: Results of the hydrolase-catalyzed cleavage of the bioemulsifier of Saccharomyces cerevisiae for the cell suspension (pH 6.6, 30 ° C).

• ¹ t _i - incubation
• ² t _p - phase separation time
• ³ without enzyme addition

The Results summarized in the table clearly show that the phase separation time by incubation with hydrolases clearly be shortened to a commercially-technically meaningful time scale can. This is a receipt for the deemulsifying influence of Hydrolases and shows that with the use of hydrolases efficiency of extraction quantity and cleanliness of the product to be isolated to be significantly increased can.

Embodiment 4 Determination the extent the enzyme-catalyzed hydrolysis

The extent of enzyme-catalyzed hydrolysis of an ethyl ester function during the erfindungsge The process for product isolation with biocatalytic deemulsification was determined using the example of ethyl 2-cyclopentanonecarboxylate for various hydrolases.

To an incubation with enzyme was carried out analogously to embodiment 3a. Instead of of culture medium was incubated in 0.5 M potassium phosphate buffer (pH 6.6) to give a meaningful liquid chromatographic To ensure analytics.

to Determination of the extent the enzyme-catalyzed hydrolysis of the ethyl ester function was exploited, that the hydrolysis product of the 2-cyclopentanonecarboxylic acid ethyl ester produced by spontaneous decarboxylation cyclopentanone, which by liquid chromatography reliable of 2-cyclopentanonecarboxylic acid ethyl ester differentiated and reproducibly quantified.

• ¹ t_i – Inkubationszeit
• ² t_p – Phasenseparationszeit
• ³ ohne Enzymzugabe
• ⁴ ++ sehr gut, + gut, o durchschnittlich, – wenig geeignet, -- ungeeignet

The ratio of ethyl 2-cyclopentanonecarboxylate to cyclopentanone was determined. The degree of hydrolysis results from the ratio of the determined concentration of cyclopentanone to the concentration of ethyl 2-cyclopentanonecarboxylate used. Table 3: Results of the hydrolysis resistance tests of the ester function (pH 6.6, 30 ° C).

• ¹ t _i - incubation
• ² t _p - phase separation time
• ³ without enzyme addition
• ⁴ ++ very good, + good, o average, - unsuitable, - unsuitable

The Hydrolysis levels observed with the enzymes used are so low, they did not significantly affect the product yield impact.

Bibliography:

• Bertau, M., Bürli, M., Hungerbühler, E., Wagner, P. (2001) A novel highly stereoselective synthesis of chiral 5- and 4,5-substituted 2-oxazolidinones. Tetrahedron: Asymmetry 12, 2103-2107.
• Davoli, P .; Forni, A .; Moretti, I .; Prati, F .; Torre, G. (1999) (R) - (1) and (S) - (2) ethyl 4,4,4-trifluoro-3-hydroxybutanoate by enantioselective Baker's yeast reduction. Enzymes Microbiol. Technol. 25, 149-152.
• Haberland, J .; Hummel, W .; Daussmann, T .; Liese, A. (2002) New continuous production process for enantiopure (2R, 5R) -hexanediol. Org. Process Res. Dev. 6, 458-462.

Claims (13)

A method for isolating xenobiotic organochemical compounds produced by biocatalytic conversion by means of yeast cells by extraction with an organic solvent from cell suspensions or after a cell separation step from cell culture media, characterized in that before extraction of the cell suspension or the cell culture medium for enzymatic degradation of excreted by the yeast cells Bioemulgatoren a hydrolase is added from at least one protease and / or glycosylase and after a subsequent incubation step, the extraction with the organic solvent. Method according to claim 1, characterized in that that the volume of organic solvent used for extraction is smaller than the volume of cell culture medium used for extraction or the cell suspension. Method according to claim 1 or 2, characterized that extraction directly from the cell suspension without a previous one Cell separation step is done. Method according to one of claims 1 to 3, characterized that the yeast cells of the genus Saccharomyces cerevisiae are a member. Method according to one of claims 1 to 4, characterized that the protease belongs to the group of serine proteases or metalloproteases belonging to is. Method according to one of claims 1 to 5, characterized the incubation with the hydrolase component is carried out at 25 ° C to 45 ° C. Method according to one of claims 1 to 6, characterized that incubation with the hydrolase component at a pH between pH 5 and pH 8 becomes. Method according to one of claims 1 to 7, characterized that the hydrolase component having an enzyme activity of preferably 10 units to 100 units per ml cell culture medium or cell suspension added becomes. Method according to one of claims 1 to 8, characterized that incubation with the hydrolase component in a period of time of up to 5 hours. Method according to one of claims 1 to 9, characterized that the Hydrolasekomponente already at the beginning of the cell culture the Medium is added. Method according to claim 10, characterized in that that while cell culture, the cell suspension or the cell culture medium continuously is incubated with the hydrolase component and continuously or semi-continuously organic components from the cell suspension or the cell culture medium be isolated by extraction. Method according to one of claims 1 to 11, characterized that the solvent used for the extraction after separation of the extracted product for further extraction becomes. Method according to claim 12, characterized in that that for continuous extraction, the solvent in a cycle guided becomes.