DE102005031536A1 - Breaking down emulsifying substances in cell suspensions or cell-free culture medium of biotransformations, comprises adding microorganisms which differ from microorganisms used for biotransformation to cell suspension or cell-free medium - Google Patents

Abstract

Breaking down emulsifying substances present in cell suspensions (CS) or in cell-free culture medium (CM) of biotransformations comprises adding microorganisms to CS or CM to break down the emulsifying substances, where these microorganisms are different from the microorganisms used for the biotransformation. Independent claims are also included for the following: (A) use of microorganisms for the break-down of emulsifying substances in CS or CM of a biotransformation; and (B) isolating the products of a biotransformation from CS or CM of a biotransformation, comprising: (a) adding microorganisms, where the microorganisms are for the break-down of the emulsifying substances and which are different from the microorganisms used for the biotransformation; and (b) extracting the product of the biotransformation, using an organic solvent, after incubating CS or CM with the emulsifying substances-decomposing microorganisms.

Description

The The invention relates to a method for degrading in cell suspensions or cell culture media containing emulsifying substances, in particular for use in the extractive isolation of organic chemical Compounds made by biotransformation. This is particularly relevant in the production of chiral intermediates for the Pharmaceutical Industry.

The biotechnological-chemical synthesis of organic chemical compounds with the help of microorganisms - the so-called "biotransformation" - is known (CSUK et al., 1991, YAMADA & SHIMIZU, 1988). In the biotransformation, the enzymatic conversion takes place a non-natural one (xenobiotic) organic chemical compound (substrate) in one others (product). Biotransformation is a chemical reaction, by a specific isolated enzyme or enzyme system or an enzyme or enzyme system present in cells is catalyzed.

He follows the enzymatic conversion by a living cell Enzyme or enzyme system, it is also called a whole-cell biotransformations. The living cells are mostly microorganisms such as. Saccharomyces cerevisiae.

The Most of such enzymes or enzyme systems are for metabolic processes and the reproduction of the cells or microorganisms necessary and serve while the whole-cell biotransformation merely as biocatalysts for the chemical Reactions (YAMADA & SHIMIZU, 1988). The catalyzed conversions often go far more specific and selective than conventional organic reactions. These Advantages combined with high space-time yields, comparatively inexpensive, renewable starting materials and often better environmental the processes have led to that the number of biotransformation processes used in the industry increase enormously.

The focus for the application of this technology lies in the production of chiral pharmaceutical intermediates. By means of whole-cell biotransformation with S. cerevisiae, for example, β-hydroxy esters are obtained by the reduction of various β-ketoesters. β-Hydroxy esters are an important precursor in the synthesis of chiral 4- and 4,5-substituted 2-oxazolidinones:

at the oxazolidinones are a group of antibiotics with a new mechanism of action, especially against gram-positive Cocci such as the methicillin-resistant Staphylococcus aureus used become. Due to the resistance development of many germs (especially in the Hospital) opposite common Antibiotics, the development of novel antibiotics is of great importance (Zurenko et al., 2001).

Further Examples are the stereoselective reduction of ethyl 2-cyclopentanonecarboxylate to ethyl (1R, 2S) -cis-2-hydroxycyclopentanecarboxylate by whole cell biotransformation with Saccharomyces cerevisiae, the stereoselective reduction of 4-Chloracetessigester by a Whole-cell biotransformation with Saccharomyces cerevisiae, the stereoselective Reduction of 4,4,4-trifluoroacetoacetate by whole cell biotransformation with Saccharomyces cerevisiae or the stereoselective reduction of acetonylacetone by whole cell biotransformation with Lactobacillus kefir.

Various Process for the treatment of biotransformation products from the broth of microorganisms are known or already known from the literature published by the inventors (Haberland et al, 2002; Bertau, 2001; Davoli et al., 1999 and references cited therein).

The Products of biotransformations are overwhelmingly used for manufacturing used pharmacological agents, with the result that these problems in product recovery not only more economical Nature, but also on the pharmacological efficacy of the final product.

In usually after completion of the whole cell biotransformation the crude product-containing culture broths (Cell suspensions) concentrated to dryness and the biotransformation products subsequently with an organic solvent extracted. Optionally, prior to concentration, a cell separation step occurs.

at This process is the biotransformation product by lipophilic Cell constituents are contaminated to a considerable degree, resulting in time-consuming cleaning operations necessary. According to the prior art is usually a thermal Purification process (distillation) applied to the desired product to separate from the lipophilic cell components.

For steam-volatile compounds become in this process depending on their Wasserdampffflüchtigkeit Partial loss rates of up to 90% observed. To low-volatility products separated by distillation from the lipophilic cell components are regular too in high vacuum high temperatures (70 ° C to 130 ° C) necessary. In these conditions It may lead to the formation of reactive by-products, which u.a. to to lead may indicate that the biotransformation product has lower product purity. It may also result in partial loss of stereoisomeric purity come. This results in up to 80% loss of the desired product. In addition, the formation of reactive species poses potential dangers in the Occupational safety area.

alternative takes place to isolate the biotransformation product from the culture broth the whole-cell biotransformation, a so-called cell separation step, i.e. the cells contained in the cell suspension are determined by Centrifugation, filtration, etc. separated. Following will be the biotransformation products with organic solvents, e.g. Ether, off the aqueous cell-free (culture) medium extracted. This usually has an up to tenfold surplus on organic solvent to the aqueous phase be added.

Problematic it is that when adding the organic solvent to the cell-free Culture medium Gel and Schleimbildungsphänomene occur. These pose a central complication in the isolation of microbially produced chiral pharmaceutical intermediates from Ganzzellbiotransformen represents and sometimes have drastic effects on the recovery rate.

in the laboratory scale can gel and slime formation by centrifugation of the mixture from watery cell-free medium and ether (Davoli et al., 1999 and cited literature). This method is already in the laboratory scale clearly dangerous and for the application on a large scale unfit.

The Gel and slime formation during extraction with organic solvents is due to the presence of emulsifying agents in the cell suspension or in cell-free culture medium after cell separation.

The Presence of emulsifying agents decreased during extraction the efficiency of the extraction regarding quantity and purity of the product to be isolated. At the same time leads the Presence of emulsifying agents to form over several Weeks or months of stable gels or mucus.

When Part of these emulsifying agents were the so-called bio-emulsifiers identified. These are surface-active substances that from those to whole-cell biotransformation used microorganisms in response to by the xenobiotic Substrates or products caused cell stress actively formed become. They are classified into glycopeptides, lipopeptides and Glycolipids.

DE 103 29 819 A1 discloses a process for the enzymatic cleavage of these bioemulsifiers by the use of hydrolases. Before extraction, a hydrolase component is added to the cell suspension. Alternatively, the addition of the hydrolase component takes place after a cell separation step to the cell-free culture medium. In a subsequent incubation step, the addition of the hydrolase serves for the enzymatic degradation of the bioemulsifiers excreted by the cells. After incubation, extraction with the organic solvent then takes place. The formation of over days to months of stable gels in the extraction with organic solutions is prevented by the hydrolytic cleavage of Bioemulgatoren.

The for the However, enzymatic deemulgation hydrolases used must be previously time and costly isolated from the cell and cleaned.

at extraction with organic solvents Phase separation times of 30 minutes are achieved when Pronase E of Streptomyces criseus is used in cell-free medium. When protease N-01 from Bacillus subtilis is used in the yeast suspension phase separation times of 60 minutes are achieved. Around a continuous extraction of the product in the mixer in the technical scale however, phase separations of maximum are to be realized 60 seconds required.

task The invention therefore provides a method of degrading in cell suspensions or in the cell-free culture medium emulsifying substances provide the phase separation times during extraction with organic solvents shortened to a commercially-technically meaningful time scale and thus allowing a continuous extraction.

According to the invention Task solved by a process for degrading emulsifying substances, which in cell suspensions or in the cell-free culture medium of whole cell biotransformations containing the cell suspension or the cell-free culture medium Microorganisms are added. The microorganisms that degrade are added to the emulsifying substances differ thereby from the microorganisms leading to the whole cell biotransformation be used.

Surprisingly It has been found that the emulsifying substances contained in cell suspensions or contained in the cell-free culture medium of whole cell biotransformations are, not only consist of the known so-called. Bioemulgatoren, but that other, unidentified emulsifying agents, which do not split by hydrolases or only incompletely in cell suspensions or in the cell-free culture medium of Biotransformations are included.

Therefore, even after the cleavage of the bioemulsifiers by hydrolases remains an emulsifying residual activity in cell suspensions or in cell-free media, which leads to that in the extraction with organic solvents after DE 103 29 819 A1 no sufficiently short phase separation times are achieved, which would allow a continuous extraction with organic solvents in the mixer settler (mixer-settler).

So far In addition to the bioemulsifiers, there were no other components in the product Cell suspensions or cell-free media are included, as emulsifying Substances known. about the nature of the species that causes the emulsifying residual activity, the is a continuous extraction in Mischabsetzer prevent is nothing known until now. Emulsifying properties of mono-, Oligo- and polysaccharides or free fatty acids, peptides or proteins could not be proven. These would also be a hydrolase-mediated Cleavage accessible. In particular, because most biomolecules, but in any case known amphiphilic biomolecules, split by hydrolases.

Surprisingly was also found to be emulsifying agents in cell suspensions or in cell-free media of whole-cell biotransformations are degraded by the addition of microorganisms. The Microorganisms that are added to break down the emulsifying substances be different from the microorganisms that contribute to Whole cell biotransformation can be used.

Next the degradation of bioemulsifiers by the hydrolase system to the de-emulsification The microorganisms used are also not affected by hydrolases degradable others still in the cell suspension or in the cell-free Culture medium containing emulsifying agents degraded. Thereby For example, the phase separation time during extraction will be advantageous far less that continuous extraction of the product - for example in the mixer-possible is.

Becomes for example, a cell suspension of Saccharomyces cerevisiae, the whole cell biotransformation was used, first subjected to a cell separation step and then the cell-free culture medium extracted with an organic solvent, so done the complete Phase separation in 20 seconds, provided the cell-free culture medium was incubated with Bacillus subtilis cells prior to extraction. Becomes the cell suspension of Saccharomyces cerevisiae, which is used for whole-cell biotransformation was used without prior cell separation step directly incubated with Bacillus subtilis followed by organic solvent extracted, the phase separation occurs within 50 to 60 Seconds.

Furthermore, the de-emulsification with microorganisms is advantageously associated with lower costs than the known de-emulsification by means of hydrolases. Microorganisms such as B. subtilis can usually be easily grown under simple conditions. Preferably, non-pathogenic microorganisms are used. While for the enzymatic de-emulsification, the hydrolases from the cell have to be isolated and purified in a time- and cost-intensive manner, the whole cells can be used in the microbial de-emulsification.

Advantageous remains due to the degradation of the invention of the emulsifying agents observed in the prior art Problem of gel and slime formation on addition of the organic extractant out. In the method according to the invention let yourself during extraction no significant conversion of lipophilic cell components prove in the organic phase.

simultaneously will after implementation the method according to the invention with a volume of organic solvent that is smaller as the volume of the aqueous Phase, observed in the subsequent extraction phase separation. From ecological and economic establish this is in the process according to the invention used volumes of solvent therefore preferably smaller than the volume used for extraction of the cell culture medium 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 on purification steps, which vary according to the state of Connect technology to the extraction, be waived. To extraction has to be done only for extraction volatile solvent in the usual way, z. B. by distillation, separated from the semi-volatile product component become. This separation can be gentle at low temperatures (25 ° C to 35 ° C) and To press (600-1030 hPa).

By the saving more complex Cleaning steps reduce the personnel and material costs and the yield of product increased. At the same time reduced by the absence of gel and slime also the amount of solvent necessary for the extraction. Is advantageous the solvent used for extraction After separation of the product so pure that it is for subsequent extractions without prior Cleaning can be used again or be recycled can. 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 Work safety significantly increased, because now also thermolabile products without the risk of formation more reactive By-products can be worked up.

Prefers these are the microorganisms that cause whole-cell biotransformation be used to yeast cells or bacteria. Especially preferred are yeasts of the genus Saccharomyces or Candida or bacteria of the genus Lactobacillus or Escherichia, especially Sacharomyces cerevisiae, Lactobacillus kefir are preferred or Escherichia coli. Algae are also for whole-cell biotransformation suitable.

The inventive method Deemulsification is not only in the processing of whole-cell biotransformations applicable, but also for the processing of biotransfomations, in which enzymes instead of microorganisms or whole cells for biocatalysis used in enzyme preparations, usually derived from microorganisms as well emulsifying agents are included in the extraction with organic solvents lead to gel and slime formation. This is especially true for technical enzyme preparations to.

The Selection of the method according to the invention for the degradation of the emulsifying substances used microorganisms is due to the microorganism used for biotransformation and determines the product to be isolated. Preferably it is These are bacteria, particularly preferably those that the Genus Bacillus, Pediococcus or Rhodococcus, especially preferred are Bacillus subtilis, Pediococcus acidilactici and Rhodococcus erythopolis.

For working up by a whole cell biotransformation with Saccharomyces cerevisiae done Reduction of ethyl acetoacetate to ethyl hydroxybutyrate, for example, is particularly suitable for incubation of the cell-free culture medium with Bacillus subtilis as a de-emulsifying microorganism.

Becomes the microorganism degrading the emulsifying agents directly - without cell separation step - the cell suspension added and incubated the microorganisms contained in the cell suspension for whole-cell biotransformation be used, the de-emulsifying microorganisms as another Use carbon and energy source, leaving the de-emulsifying Properties of the microorganisms degrading the emulsifying agents be reduced.

In a preferred embodiment Therefore, the incubation with the emulsifying agents degrading microorganisms not with the cell suspension, but with the cell-free culture medium after a cell separation step, e.g. centrifugation or filtration.

The Incubation with the microorganisms that degrade the emulsifying Agents are added, is preferably carried out under conditions which increase the growth of emulsifying agents added Reduce microorganisms. This will make the synthesis of your own, disturbing Bioemulgatoren or emulsifying agents by those used for de-emulsification Microorganisms are reduced and yet the existing emulsifying Agents are used as the sole source of carbon and energy.

Becomes Bacillus subtilis as a microorganism to degrade the emulsifying Agents used, the incubation is preferably carried out under oxygen-limited conditions. For the creation of oxygen-limited Conditions become the incubation vessels, for example, airtight completed, leaving only the remaining oxygen above the Medium can be used.

In a preferred embodiment incubation of the cell-free medium with B. subtilis is done in such a way that the concentration of dry weight B. subtilis in cell-free medium between 0.04 and 0.06% (w / v). The incubation is preferred at a temperature of 25 ° C up to 37 ° C, especially preferred at 30 ° C and a pH between pH 6 and pH 7, particularly preferred between pH 6.4 and 6.8.

Become Pediococcus acidilactici or Rhodococcus erythropolis as microorganisms used to degrade the emulsifying agents, the incubation takes place preferably under aerobic conditions. For the creation of an aerobic Milieus were the vessels below Sterile conditions suitably closed air permeable. It showed Pediococcus acidilactici and Rhodococcus erythropolis a complete Phase separation after 80 s or 180 s.

To Termination of the incubation with the degradation of the emulsifying substances used microorganisms is preferably carried out directly the extraction with the organic solvent, preferably with tert-butyl methyl ether.

By the following figures and embodiments will be the invention explained in more detail:

Embodiment 1

Deemulsification of cell-free culture medium

biotransformation of acetoacetic acid ethyl ester with Saccharomyces cerevisiae

13% (w / v) S. cerevisiae (cube yeast) will be in a solution consisting of 10% (w / v) sucrose and 1% (w / v) calcium carbonate and tap water suspended. After a growth phase of 30 min under aerobic conditions at 30 ° C and a stirring speed of 100 U / min, the ethyl acetoacetate over a Period of 44 h added. After completion of the dosing is the culture broth under the same conditions for further cultured for a further 4 h to complete reduction of ethyl acetoacetate to hydroxybutyric acid ethyl ester to grant.

preculture of Bacillus subtilis

For the preparation of the precultures is a separation smear of B. subtilis on yeast nutrient Ion agar prepared and cultured for 48 h at 30 ° C. From the overgrown agar plate, a single colony is transferred to the preculture, a yeast broth. The preculture is cultivated at 30 ° C and 150 U / min until the optical density at 600 nm corresponds to 0.8 absorbance units.

Incubation of the cell-free Medium with B. subtilis

The broth is for each 20 min at 3000 × g and 20000 × g centrifuged to completely separate the yeast cells. For the incubation of the cell-free medium becomes 1.6 times the volume of preculture B. subtilis at 3000 × g centrifuged. The cells are harvested and the cell-free medium so that the concentration of dry weight B. subtilis in the cell-free medium is 0.044% (w / v). The incubation of the cell-free Medium with B. subtilis is oxygen-limited for 48 h at 30 ° C and 100 rpm. For To create oxygen-limited conditions, the incubation vessels become airtight completed, leaving only the remaining oxygen above the Medium can be used. Under these conditions is growth the bacilli and thus the synthesis of their own, disturbing Bioemulgatoren reduced, and yet the yeast's bio-emulsifiers are the only ones Carbon and energy source recycled.

workup the incubation broth by extraction

To Termination of the incubation is the incubation broth directly by three times Extraction with tert. Butyl methyl ether worked up, the organic Phase over Dried magnesium sulfate and the solvent removed under vacuum. The phase separation after incubation with B. subtilis is after 20 s completely.

Embodiment 2:

Deemulsification of a cell suspension

The Biotransformation of ethyl acetoacetate with Saccharomyces cerevisiae and the preculture of Bacillus subtilis take place analogously to the embodiment 1. The Bacillus subtilis cells are harvested and added directly to the S. cerevisiae cell suspension. The incubation and the work-up of the incubation broth by Extraction carried out analogously to the embodiment 1. Phase separation after incubation with B. subtilis is after 50 to 60 s complete.

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-hydroxy butanoate by enantioselective Baker's yeast reduction. Enzyme Microb. Technol. 25, 149-152.
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Claims (11)

Process for the degradation of emulsifying substances contained in cell suspensions or in the cell-free culture medium of biotransformations, characterized in that microorganisms are added to the cell suspension or the cell-free culture medium for degrading the emulsifying substances, wherein the microorganisms added to degrade the emulsifying substances differ from the microorganisms used for biotransformation. Method according to claim 1, characterized in that that the microorganisms used for biotransformation Yeast cells, algae or bacteria are. Method according to claim 2, characterized in that that the yeast cells used for biotransformation belonging to the genus Saccharomyces or Candida or that the bacteria, which are used for whole-cell biotransformation, the genus Lactobacillus or Escherichia belong. Method according to one of claims 1 to 3, characterized that the microorganisms that degrade the emulsifying substances be used, bacteria are. Method according to claim 4, characterized in that that the bacteria used to break down the emulsifying substances belonging to the genus Bacillus, Pediococcus or Rhodococcus. Method according to claim 5, characterized in that that the bacteria used to break down the emulsifying substances be selected are from Bacillus subtilis, Pediococcus acidilactici and Rhodococcus erythopolis. Use of microorganisms to degrade emulsifying Substances in cell suspensions or in the cell-free culture medium of Biotransformations. Use according to claim 7, characterized that the microorganisms that degrade the emulsifying substances be used, bacteria are. Use according to claim 8, characterized that the bacteria used to break down the emulsifying substances belonging to the genus Bacillus, Pediococcus or Rhodococcus. Use according to claim 9, characterized that the bacteria used to break down the emulsifying substances be selected are from Bacillus subtilis, Pediococcus acidilactici and Rhodococcus erythopolis. Process for the isolation of products of a biotransformation from a cell suspension or cell-free culture medium Biotransformation, characterized in that a. for degradation emulsifying substances of the cell suspension or the cell-free Culture medium microorganisms are added, the microorganisms, which are added to the degradation of the emulsifying substances themselves of the microorganisms used for biotransformation, distinguish and b. the product of biotransformation incubation of the cell suspension or the cell-free culture medium with the microorganisms degrading the emulsifying substances with an organic solvent is extracted.