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    • 专利摘要:
    process for removing an organic compound from an aqueous solution and reaction mixture comprising an aqueous solution and a hydrophobic organic solution. The present invention relates to a process for removing an organic compound that has one or more positive charges from an aqueous solution, comprising the steps: a) preparation of the aqueous solution containing the organic compound and a hydrophobic organic solution, which comprises a liquid cation exchanger, wherein the liquid cation exchanger is hydrophobic, and wherein the liquid cation exchanger has one or more negative charges and an overall negative charge, b) bringing the aqueous solution into contact with the organic solution, and c ) separating the organic solution from the aqueous solution.
    公开号:BR112013020912B1
    申请号:R112013020912-7
    申请日:2011-12-01
    公开日:2021-06-01
    发明作者:Frank Erhardt；Thomas Haas；Martin Roos；Daniel Demicoli；Markus POTTER；Anja Schubert；Jan Christoph Pfeffer；Thomas Tacke；Harald Hãger；Andreas Pfennig；Marie-Dominique Przybylski
    申请人:Evonik Operations Gmbh；
    IPC主号:
    专利说明:

    [0001] The present invention relates to a process for removing an organic compound that has one or more positive charges from an aqueous solution, comprising the steps: (a) preparation of the aqueous solution containing the organic compound and a hydrophobic organic solution, which comprises a liquid cation exchanger, the liquid cation exchanger being hydrophobic, (b) bringing the aqueous solution into contact with the organic solution and (c) separating the organic solution from the aqueous solution, in the case of the organic compound it is a compound of the formula NH3+-A-COOR1 as well as related reaction mixtures.
    [0002] A fundamental problem in the biotechnological production of fine chemicals from renewable raw materials, which are traditionally synthesized from fossil fuels, is to convert the product once obtained, which is typically present in a large-volume aqueous phase, into an organic phase. This conversion is carried out, on the one hand, to concentrate a ready-made intermediate or final product and to optionally allow synthetic processing in subsequent reaction steps in organic solution, on the other hand, to improve the reaction yield in aqueous phase through removing the desired product or, at all, first allowing the reaction to proceed within a technically suitable scope. The direct thermal concentration of the product often present in low concentrations of the large volume aqueous solution is, as a rule, not adequate.
    [0003] The distribution of a compound in a two-phase system comprises a hydrophilic, aqueous phase and a hydrophobic, organic phase, which do not mix, depends significantly on the physicochemical properties of the respective compound. While compounds with a high proportion of hydrocarbons or consisting exclusively of unsubstituted hydrocarbons are preponderantly enriched in the hydrophobic phase, the compound with a high proportion of polar groups, such as functionalities containing heteroatoms, and very particularly, charged compounds are preponderantly or practically exclusively present in the aqueous phase, which makes conversion to an organic phase difficult.
    [0004] The distribution of a compound in the two-phase system mentioned after adjusting the equilibrium, is often described with the aid of distribution coefficients, for example, according to the equation of Nernstα = cphase 1/Cphase 2.
    [0005] A special distribution coefficient is Kow, also called P-value, which characterizes the distribution equilibrium of a compound between an octanol phase and an aqueous phase: Kow = P = Coctanol/Water
    [0006] An example of a positively charged organic compound, industrially much sought after represents the 12-aminolauric acid (ALS) and its derivatives, in particular, the methyl ester (ALSME). ALS is an important starting material in the production of polymers, eg for the preparation of piping and nylon systems. Traditionally, ALS is produced from fossil raw materials in a low-yield process using laurinlactam, which is synthesized through the trimerization of butadiene, subsequent hydrogenation with formation of cyclododecane, subsequent oxidation to cyclododecanone, reaction with hydroxylaurin and subsequent molecular rearrangement of Beckmann. A promising path for the biotechnological production of ALS or ALSME is described in DE 10200710060705.
    [0007] The state of the art teaches obtaining positively charged organic compounds through contacting an aqueous reaction mixture comprising a biological agent with an organic phase comprising an organic solvent. Thus, DE10200710060705 describes, for example, obtaining the ALSME product from an aqueous reaction mixture by stirring with acetic acid ethyl ester. Asano et al. (2008) publish the extraction of ALS with toluene from an aqueous reaction solution comprising an enzyme that synthesizes ALS.
    [0008] Therefore, the objective underlying the present invention is to develop a process for the removal of positively charged organic compounds, particularly w-aminocarboxylic acids, with at least one positive charge from an aqueous reaction mixture, a charge being desired the most advantageous possible distribution balance between the reaction mixture and a hydrophobic organic phase used as extracting agent, i.e. the distribution balance should, as far as possible, be on the side of the hydrophobic organic phase.
    [0009] Another objective underlying the invention is to develop a process for removing organic compounds with at least one positive charge, particularly w-aminocarboxylic acids, from an aqueous solution comprising a biological agent using a hydrophobic organic phase as an agent extraction, in which the distribution equilibrium is, as far as possible, on the side of the hydrophobic organic phase.
    [00010] Another objective underlying the invention is to develop a process for removing organic compounds with at least one positive charge, particularly w-aminocarboxylic acids, from an aqueous solution using a hydrophobic organic solution as an extracting agent, that harms as little as possible or retards the growth of biotechnologically relevant microorganisms, in particular, Escherichia coli and/or in this case, reduces as little as possible the number of cells capable of dividing and/or viable and/or respiratory active and/or metabolically and synthetically active.
    [00011] Finally, the objective underlying the invention is to discover a process for removing an organic compound with at least one positive charge, particularly w-aminocarboxylic acids, from an aqueous solution comprising a biological agent using a hydrophobic organic phase as an extraction agent, in which the totality of the decisive properties for the yield, the total conversion and rapid feasibility of a biotechnological synthesis process that serves as a basis, in particular, the toxicity of the organic phase in relation to the biological agent and the absorption of compound in the organic extracting agent, with respect to the total yield or a faster course or, in the case of a continuous process, a maximum service life of the biological agent is optimized, particularly in the case where the organic compound with at least one charge positive represents the product or an intermediate product of the synthesis process, which is synthesized with the participation of a catalytic activity of the agent and biological.
    [00012] This and other objectives are solved by the objective of the present application and, in particular, also by the objective of the attached independent claims, the forms of implementation resulting from the sub-claims.
    [00013] The objective underlying the invention is solved in a first aspect by a process for the removal of an organic compound that has one or more positive charges from an aqueous solution, comprising the steps (a) preparation of the aqueous solution containing the organic compound and a hydrophobic organic solution, comprising a liquid cation exchanger, the liquid cation exchanger being hydrophobic, (b) contacting the aqueous solution with the organic solution, and (c) separating the organic solution of the aqueous solution, and in the case of the organic compound it is a compound of the Formula INH3+-A-COOR1 (I), where R1 is hydrogen, methyl, ethyl or a negative charge and A is a straight-chain alkylene group, unsubstituted with at least three, preferably with at least eight carbon atoms, and in the case of the liquid cation exchanger it is a fatty acid.
    [00014] In a first embodiment of the first aspect, the problem is solved by a process according to one of claims 1, with the temperature in step (b) being 28 to 70, preferably 30 to 37oC.
    [00015] In a second embodiment, which is also an embodiment of the first embodiment of the first aspect, the problem is solved by a process according to one of claims 1 to 2, with the pH value in the step (b) amounts to 6 to 8, preferably 6.2 to 7.2.
    [00016] In a third embodiment, which is also an embodiment of the first to the second embodiment of the first aspect, the problem is solved by a process, in which the ratio of material quantity of the liquid cation exchanger for organic compost it matters at least 1.
    [00017] In a third embodiment, which is also a embodiment of the first to the third embodiment of the first aspect, the problem is solved by a process, in which the volumetric ratio of the organic solution to the aqueous solution matters in 1:10 to 10:1.
    [00018] In a fourth embodiment, which is also an embodiment of the first to the third embodiment of the first aspect, the problem is solved by a process, in which the liquid cation exchanger is a fatty acid with more of 12, preferably with 14 to 22, even more preferably, 16 to 18 carbon atoms.
    [00019] In a fifth embodiment, which is also an embodiment of the first to the fourth embodiment of the first aspect, the problem is solved by a process, in which the liquid cation exchanger is an unsaturated fatty acid, preferably oleic acid or erucic acid.
    [00020] In a sixth embodiment, which is also an embodiment of the first to the fifth embodiment of the first aspect, the problem is solved by a process, in which the aqueous solution further comprises a biological agent with catalytic activity.
    [00021] In a seventh embodiment, which is also an embodiment of the first to the sixth embodiment of the first aspect, the problem is solved by a process, in which the biological agent presents a cell, preferably a bacterial cell and the cell even more preferably is a recombinant alkanemonooxygenase, a recombinant transaminase, as well as preferably, in addition, at least one enzyme from the group, which comprises an alcohol dehydrogenase, an alanine dehydrogenase and the gene product AlkL or variants of that.
    [00022] In an eighth form of embodiment, which is also a form of embodiment from the first to the seventh form of embodiment of the first aspect, the problem is solved by a process, in which the presence of the organic compound acts disadvantageously on the catalytic activity, preferably due to the fact that, in the case of the organic compound, it is a toxic compound for the cell.
    [00023] In a ninth embodiment, which is also a embodiment of the first to the eighth embodiment of the first aspect, the problem is solved by a process, in which the organic solution contains, in addition, at least one organic solvent, preferably a fatty acid and/or a fatty acid ester.
    [00024] In a tenth embodiment, which is also an embodiment of the first to the ninth embodiment of the first aspect, the problem is solved by a process according to claim 12, in which the organic solution comprises as liquid cation exchanger, 20 to 80% by volume, preferably 25 to 75% by volume, of oleic acid and as a solvent, lauric acid methyl ester and in the case of the organic compound it is 12-aminolauric acid methyl ester and in the aqueous solution is present a bacterial cell, which has a recombinant alkanemonooxygenase, a recombinant transaminase, as well as preferably, in addition, at least one enzyme from the group, which comprises an alcohol dehydrogenase, an alanine dehydrogenase and the gene product AlkL or its variants.
    [00025] In a second aspect, the problem underlying the invention is solved by a reaction mixture comprising an aqueous solution and a hydrophobic organic solution, wherein the hydrophobic organic solution comprises a fatty acid, more preferably a fatty acid with more than 12 carbon atoms, even more preferably an unsaturated fatty acid as a liquid cation exchanger and in the case of the aqueous solution it is a compound of the Formula (I)NH3+-A-COOR1 (I)where R1 is hydrogen, methyl, ethyl or a negative charge and A is a straight chain, unsubstituted alkylene group with at least three, preferably at least eight carbon atoms.
    [00026] In an embodiment of the second aspect, the problem underlying the invention is solved by a reaction mixture according to the first aspect, in which the aqueous solution further comprises a cell, which has a recombinant alkanemonooxygenase, a recombinant transaminase, as well as preferably furthermore at least one enzyme from the group comprising an alcohol dehydrogenase, an alanine dehydrogenase and the AlkL gene product or variants thereof.
    [00027] Other embodiments of the second aspect comprise all embodiments of the first aspect.
    [00028] The objective underlying the invention is solved in a fourth aspect by a process for the removal of an organic compound, which has one or more positive charges, from a solution, comprising the steps: (a) preparation of the solution aqueous containing the organic compound and a hydrophobic organic solution, comprising a liquid cation exchanger, the liquid cation exchanger being hydrophobic and the liquid cation exchanger having one or more negative charges and a total negative charge,(b ) contacting the aqueous solution with the organic solution, and (c) separating the organic solution from the aqueous solution.
    [00029] In a second embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first embodiment of the present invention, the process comprises the step: (d) processing the organic solution, preferably through re -extraction of the organic compound into another aqueous solution.
    [00030] In a third embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the second embodiment of the present invention, the temperature in step (b) of the process according to the invention matters at 28 to 70oC, preferably 30 to 37oC.
    [00031] In a fourth embodiment of the fourth aspect of the present invention, which also represents a form of embodiment of the first to the third embodiment of the present invention, the pH value in step (b) of the process according to the invention it amounts to 3 to 8, preferably 6 to 8, particularly preferably 6.2 to 7.2.
    [00032] In a fifth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the fourth embodiment of the present invention, the ratio of the amount of liquid cation exchanger material to organic compound in the process matters in at least 1.
    [00033] In a sixth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the fifth embodiment of the present invention, the volumetric ratio of the organic solution to the aqueous solution matters in 1:10 to 10:1.
    [00034] In a seventh embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the sixth embodiment of the present invention, the organic compound has at least one positively charged substituent of Formula (I) -N+R2R3R4 (I) or, provided that at least one substituent from the group comprising R2, R3 and R4 is hydrogen, has the non-protonized form thereof, wherein R2, R3 and R4 independently are selected from the group comprising hydrogen, methyl , ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxy, substituted or unsubstituted alkyl or alkenyl and/or straight or branched or cyclic.
    [00035] In an eighth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the seventh embodiment of the present invention, the organic compound has the Formula (II)ZA-N+R2R3R4 ( II)or, provided that at least one substituent from the group comprising R2, R3 and R4 is hydrogen, it has the non-protonized form thereof, wherein R2, R3 and R4 independently of one another are selected from the group consisting of hydrogen, methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxy, substituted or unsubstituted alkyl or alkenyl and/or straight or branched or cyclic, where A represents a hydrocarbon chain comprising at least three carbon atoms, preferably an unsubstituted alkenyl group wherein Z is selected from the group, comprising -COOH, -COOR5, -COH, -CH2OH and non-protonized forms thereof, wherein R5 is selected from the group. endohydrogen, methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxy, substituted or unsubstituted alkyl or alkenyl and/or straight or branched or cyclic.
    [00036] In a ninth embodiment of the fourth aspect of the present invention, which also represents a form of embodiment of the first to the eighth embodiment of the present invention, the organic compound has the Formula III NH3+-A-COOR1 (III), or a non-protonized form thereof, wherein R 1 is hydrogen, methyl or ethyl and A is an unsubstituted straight chain alkylene group having at least three carbon atoms.
    [00037] In a tenth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the ninth embodiment of the present invention, the liquid cation exchanger has at least one alkyl or alkenyl group with at least minus six carbon atoms, as well as a terminal substituent of the group, which comprises -COOH, -OSO2H, -OPO(OH)2- and -OPO(OH)O- and non-protonized forms thereof.
    [00038] In an eleventh embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the tenth embodiment of the present invention, the liquid cation exchanger is an unsaturated fatty acid, preferably oleic acid .
    [00039] In a twelfth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the eleventh embodiment of the present invention, the aqueous solution further comprises a biological agent with catalytic activity.
    [00040] In a thirteenth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the twelfth embodiment of the present invention, the biological agent is a cell, preferably a bacterial cell.
    [00041] In a fourteenth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the thirteenth embodiment of the present invention, the presence of the organic compound acts disadvantageously on the activity catalytic.
    [00042] In a fifteenth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the fourteenth embodiment of the present invention, the organic solution contains, in addition, at least one solvent organic, preferably a fatty acid and/or a fatty acid ester.
    [00043] In a sixteenth embodiment of the fourth aspect of the present invention, which also represents an embodiment of the first to the fifteenth embodiment of the present invention, the organic solution comprises as a liquid cation exchanger, 20 to 80% by volume, preferably 25 to 75% by volume, of oleic acid and as a solvent, lauric acid methyl ester and in the case of the organic compound it is 12-aminolauric acid methyl ester and a cell is present in the aqueous solution. bacterial, which has a catalytic activity participating in the synthesis of the 12-aminolauric acid methyl ester.
    [00044] In a fifth aspect, the objective underlying the invention is solved by a bioreactor comprising an aqueous solution comprising a biological agent and a hydrophobic organic solution comprising a liquid cation exchanger. In a preferred embodiment of the present invention, by the term "bioreactor" as used herein is meant any container in which biotechnologically useful microorganisms can be cultivated under controlled conditions and/or a biotechnological process can be used. , preferably the synthesis of an organic compound.
    [00045] In a second embodiment of the fifth aspect, in which case it is also an embodiment of the first embodiment of the third aspect of the present invention, the liquid cation exchanger is a fatty acid, preferably oleic acid .
    [00046] In a third embodiment of the fifth aspect, in which case it is also an embodiment of the first to the second embodiment of the third aspect of the present invention, the hydrophobic organic solution further comprises a fatty acid ester, preferably lauric acid methyl ester.
    [00047] In a fourth embodiment of the fifth aspect, in which case it is also an embodiment of the first to the third embodiment of the second aspect of the present invention, the hydrophobic organic solution comprises as a liquid cation exchanger oleic acid and as solvent, 25 to 75% by volume, of lauric acid methyl ester.
    [00048] In a fifth embodiment, in which case it is also an embodiment of the first to the fourth embodiment of the fifth aspect of the present invention, it is in the case of the organic compound, a compound of according to one of the embodiments of the first aspect of the invention.
    [00049] In a sixth aspect, the objective underlying the present invention is solved by a process for the preparation of an organic compound with one or more positive charges, in which the organic compound is toxic to cells, comprising the cultivation in an aqueous solution of cells participating in the synthesis of the organic compound, preferably cells, which catalyze at least one step of the synthesis, in the presence of a hydrophobic organic solution comprising a liquid cation exchanger and optionally an organic solvent.
    [00050] In a second embodiment of the sixth aspect of the present invention, in the case of the organic compound it is 12-aminolauric acid or 12-aminolauric acid methyl ester, in the case of the organic solvent, acid methyl ester lauric.
    [00051] Other embodiments of the fourth, fifth and sixth aspects comprise all embodiments of the first and second aspects of the present invention.
    [00052] The inventors of the present invention have found, that the efficiency of removing an organic compound with one or more positive charges from an aqueous solution to a hydrophobic organic solution can be surprisingly increased, when that organic solution comprises a liquid cation exchanger. Without wishing to be bound by any theory, the inventors of the present invention assume that the negative charge or negative charges of the liquid cation exchanger ionically interact/interact with a positive charge or with the various positive charges of organic compounds and that this interaction leads to a masking of at least one positive charge, which increases the solubility in the organic phase.
    [00053] In a preferred embodiment, the term "liquid cation exchanger", as used herein, means a compound soluble in a hydrophobic organic solvent, which, due to one or more permanent negative charges, is capable of forming a ionic reciprocal effect with at least one cation. Typically, a liquid cation exchanger comprises at least one saturated or unsaturated hydrocarbon chain, which may be straight or branched, as well as a charged negative group, for example, a carboxyl group. In a preferred embodiment, the liquid ion exchanger is a fatty acid, in a still preferred embodiment an unsaturated fatty acid, eg oleic acid. In a preferred embodiment, the liquid ion exchanger is di(2-ethylhexyl) phosphoric acid (also known as DEHPA or D2EHPA).
    [00054] In a preferred embodiment, the liquid ion exchanger has not only a total negative charge, but no positive charge. In a preferred embodiment, the term "total charge" of the ion exchanger or other molecule, as used herein, is to be understood as the sum of the charges of all functional groups covalently bonded to the molecule. For example, lauric acid at pH 7 has a negative charge as the total charge, regardless of the presence of other molecules or counterions, such as potassium ions, that are present in the aqueous solution.
    [00055] In a preferred embodiment of the present invention, it is understood by the term "putting in contact", as used herein, that two phases are directly exposed to each other and, in particular, without intermediate connection of a physical barrier , such as a membrane. Contacting is effected in the simplest case by the fact that the two phases are placed in the same container and are mixed with each other in a suitable way, for example by stirring.
    [00056] In a preferred embodiment, the organic compound has a total positive charge. In another preferred embodiment, the organic compound has no negative charges. In a preferred embodiment, the organic compound is a w-aminocarboxylic acid.
    [00057] In a preferred embodiment, the term "bears a positive charge", as used herein, means that a compound so designated presents a corresponding charge in aqueous solution with pH 0 to 14, preferably 2 to 12, 2 at 6, 8 to 12, 3 to 10, 6 to 8, more preferably at pH 7. In a preferred embodiment this is a permanently present filler. In another preferred embodiment, the term "bears a charge", as used herein, means that the corresponding functional group or compound at pH 7 is preponderantly present with the corresponding charge, i.e. at least 50, more preferably 90, even more preferably 99%.
    [00058] In a preferred embodiment of the invention, the term "containing" is to be understood in the sense of "comprising", that is, not definitive. A mixture containing A may have, in this sense, in addition to A, other components. The formulation "one or more charges" means at least one charge of a corresponding nature.
    [00059] In a preferred embodiment, by the term "hydrophobic" as used herein is meant the property of a liquid, in the presence of an aqueous phase, to form a liquid phase of its own, clearly limited by the aqueous phase. In the case of the latter, it may be a related liquid phase or an emulsion. In another preferred embodiment, the term "hydrophobic" as used herein is understood to mean the property of a compound not to essentially dissolve in water. Finally, the term in another preferred embodiment as used herein is understood such that the compound so designated has a P value (J. Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, Vol. 2 of Wiley Series in Solution Chemistry, John Wiley & Sons, Chichester, 1997), whose decadic log is greater than 0, more preferably greater than 0.5, even more preferably greater than 1 and most preferably greater than 2 .
    [00060] In another embodiment of the present invention, the liquid ion exchanger does not present or has only a moderate toxic effect on biotechnologically relevant microorganisms. By the term "toxic effect", as used herein, is meant in a preferred embodiment of the invention, the property of a compound, in contact with corresponding microorganisms, to decrease its growth rate, decrease its metabolic rate, increase its consumption, decrease its optical density or number of cells capable of growing and/or directly lead to its death and lysis. In another preferred embodiment, at least one of these effects on a toxic compound is already obtained at a low concentration, preferably at a concentration of 1000, more preferably 100, even more preferably 50 or 25, most preferably 5 mg/L. The technician knows a number of processes that can be routinely applied, through which toxicity can be investigated. These include, for example, measuring the respiration of corresponding microorganisms using O2 electrodes or the plating of comparable microorganism samples and the subsequent counting of colony forming units (cfus). In a preferred embodiment, a "moderate toxic effect" is understood to mean that microorganisms that are in a growth phase continue to grow in the presence of the compound and/or are metabolically active, however, to a lesser degree than in a control, which is incubated under the same conditions in the absence of the corresponding compound and/or have an extended lag phase.
    [00061] The contact of the aqueous and organic solution takes place under suitable conditions and, in particular, for a period of time, which is sufficient for the passage of the organic compound from the aqueous phase to the organic phase, ideally, even for the adjustment of the corresponding balance. This time and conditions can be determined by the technician as part of routine trials.
    [00062] In a particularly preferred embodiment, in the case of an organic compound that has one or more positive charges, it is an amino fatty acid in terminal position, particularly preferably, 12-aminolauric acid or a ester of that or a mixture of both compounds. The skilled person will recognize, that an ester of a fatty acid in the presence of a biological system comprising esterase activities may be partially present in the corresponding acid form and both compounds in this context should be considered as being equivalent. Therefore, fatty acids or fatty acid derivatives comprise in a particularly preferred embodiment, as used herein, also the corresponding esters, preferably methyl ester and vice versa.
    [00063] In a particularly preferred embodiment, the term "alkylene group" as used herein is a group of the Formula -(CH2)n-, i.e. an alkane with two substituents left. open, preferably terminal substituents. In the case of the two substituents, they can be, for example, an amine group and a carboxy group. In a preferred embodiment, n amounts to at least 3, even more preferably at least 6, even more preferably 11. In a "substituted alkylene chain" at least one hydrogen atom is replaced by another substituent in addition to one. hydrogen atom or an alkyl radical, preferably by an atom other than a hydrogen atom. In a particular form of embodiment, the term "unsubstituted alkylene group", as used herein, instead means a hydrocarbon chain of the Formula -(CH 2 ) n - without such a substituent.
    [00064] The temperature in step (b) does not only depend on the properties of the liquid cation exchanger, but, in particular, in the case of the contact of the aqueous and the organic solution taking place during the reaction in the aqueous phase, also on the temperature requirements of reactions optionally carried out in the aqueous phase. In particular, in the case where a biological agent such as a living cell is catalytically active in the aqueous phase, the temperature to maintain this activity must be adequate. In a preferred embodiment, the temperature in step (b) ranges from 0 to 100oC, more preferably 20 to 80oC, 28 to 70oC, 30 to 37oC, 35 to 40oC.
    [00065] The pH value in step (b) must also take into account the requirements of reactions that optionally run at the same time, the stability of educts, products, intermediates or agents. In a preferred embodiment, the pH value ranges from 3 to 8, more preferably from 6 to 8, even more preferably from 6.2 to 7.2.
    [00066] To convert the organic compound from the aqueous phase as completely as possible into the organic, a satisfactory amount of liquid cation exchanger is required. In a preferred embodiment of the present invention, the quantitative ratio of substances of the liquid cation exchanger and organic compound in at least one step, in a continuous process added to the entire course of the reaction, amounts to at least 1, that is, for each molecule of the organic compound uses at least one liquid cation exchanger molecule. In an even more preferred embodiment, the ratio is greater than 2, 3, 5, 10, 15 or 20, preferably 1.5 to 3.
    [00067] The volumetric ratio of organic solution to aqueous solution is, together with the quantitative ratio of cation exchanger substances/organic compound, is significant for an efficient process. In a particular embodiment, this amounts to 100:1 to 1:100, more preferably 20:1 to 1:20, even more preferably 10:1 to 1:10, 4:1 to 1:4, 3: 1 to 1:3 or more preferably 1:2 to 2:1.
    [00068] In a preferred embodiment of the present invention, a fatty acid is used as liquid cation exchanger. In a preferred embodiment of the present invention, by the term "fatty acid" as used herein is meant a carboxylic acid, preferably alkanoic acid, with at least 6, preferably 8, even more preferably 10, more preferably with 12 carbon atoms. In a preferred embodiment these are saturated fatty acids, in another embodiment branched. In a preferred embodiment, these are saturated fatty acids. In a particularly preferred embodiment, these are unsaturated. In another preferred embodiment, it is a straight-chain fatty acid, with at least 12 carbon atoms, comprising a double bond, preferably in position 9. In another preferred embodiment, it is a fatty acid once unsaturated, in which the double bond is in position 9 and/or 11. In another preferred embodiment, the liquid cation exchanger is an unsaturated fatty acid selected from the group comprising oleic acid, palmitoleic acid and gadoleic acid and icosenic acid. In the most preferred embodiment, this is oleic acid. In a particularly preferred embodiment, it is a fatty acid with 6, 7, 8 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30 carbon atoms, preferably with more than 12, more preferably with more than 14 carbon atoms, more preferably with 14 to 28, 14 to 22, most preferably 16 to 18 carbon atoms.
    [00069] In another preferred embodiment, a mixture of different fatty acids is used as liquid ion exchanger, such as is present, for example, in the form of soybean oil or thistle oil of spherical flowers. The mixture comprises, if necessary, a pre-hydrolysis, if the fatty acids are present as an ester.
    [00070] In a particularly preferred embodiment of the present invention, a combination of two liquid cation exchangers is used, preferably at least one of these being fatty acid.
    [00071] A particular advantage of the present invention is the compatibility of the process according to the invention with biotechnological processes and biological agents used in this case. In a particular embodiment of the present invention, by the term "biological agent with catalytic activity" as used herein is meant a biocatalyst synthesized by a cell in all purification steps, from the entire cell to the isolated molecule. In a preferred embodiment, it is a cell expressing enzymes with catalytic activity. In the case of the cell, it may be a prokaryote, including Archeans, or a eukaryote, preferably from the group comprising Pseudomonas, Corynebacterium and E. coli. In an even more preferred embodiment, the agent is a bacterial cell, even more preferably a gram-negative bacterial cell, most preferably an E. coli. In another preferred embodiment, it is a eukaryotic cell, more preferably a fungal cell, even more preferably a yeast cell, more preferably a Saccharomyces or Candida, Pichia, in particular Candida tropicalis cell. The term "cell", and a particular embodiment, is used in this application synonymously and interchangeably with the term "microorganism". Furthermore, in the case of the cell, it may be an isolated cell or a mixture of cultures.
    [00072] The cell used as a biological agent may be able to live or it may be a preparation thereof, for example, a membrane fraction or cytosolic fraction or a crude extract of the cell.
    [00073] If in the case of the biological agent it is an isolated molecule in different stages of purification, then this can be all active catalytic molecules, prepared by a cell. In a particularly preferred embodiment, it is a molecule from the group comprising peptides, carbohydrates, nucleic acids or mixed forms thereof. In a still more preferred embodiment, it is a catalytically active polypeptide. In another preferred embodiment, it is an immobilized molecule.
    [00074] The catalytic functions required for synthetic biotechnological processes are multiple. In a preferred embodiment, the term "catalytic activity" as used herein is a synthetic activity, that is, the catalysis of chemical reactions comprising the formation of at least one new covalent bond. In another preferred embodiment, it is a transport activity, that is, the ability of a molecule to carry out the transport of another molecule from one compartment to another, for example, the absorption of a substance from the aqueous medium through a cell membrane into the cell.
    [00075] In a particularly preferred embodiment, in the case of the biological agent it is a living cell, which in the presence of the liquid cation exchanger is used for catalysis, preferably to synthesize an organic compound with one or more charges positive, which subsequently or at the same time, is removed by means of the liquid cation exchanger, to the hydrophobic organic phase.
    [00076] In a particularly preferred embodiment, the presence of the organic compound acts disadvantageously on the catalytic activity. In one embodiment, this can reduce the amount of activity present, which can be expressed in the sense of a smaller kcat of an enzyme. In another embodiment, the affinity of the agent that exhibits catalytic activity can be referred to in the sense of a high KM of an enzyme. In another embodiment, the specificity of the catalytic activity can be modified, for example, so that it preferentially reacts or reacts with a substrate molecule other than the desired one. In another embodiment, the organic compound has a toxic effect on a cell as a biological agent.
    [00077] In another embodiment, in the case of the organic compound it is an organic compound, which reduces the availability of a co-substrate or an essential coenzyme. This can be, for example, the case if the organic compound inhibits a corresponding regeneration reaction.
    [00078] In addition to the liquid cation exchanger, the hydrophobic organic phase may also contain a hydrophobic solvent. This can serve to increase the absorption capacity of a liquid cation exchanger in the hydrophobic phase and prevent unwanted behavior, eg flocculation. In a preferred embodiment, the solvent is an educt of a reaction that takes place in the aqueous solution, more preferably of the substrate of an enzymatic-catalytic reaction that takes place in the aqueous solution. In a preferred embodiment, it is a fatty acid ester. In a particularly preferred embodiment, the solvent is a fatty acid ester, preferably a fatty acid methyl ester, of a fatty acid, which serves as a liquid cation exchanger.
    [00079] The proportion of the solvent, if present, in the hydrophobic organic phase is, in a preferred embodiment, 1 to 99 percent by volume (% by volume). In a preferred embodiment, the proportion of solvent amounts from 10 to 90, more preferably from 20 to 80, even more preferably from 25 to 75% by volume.
    [00080] In a more preferred embodiment of the process, in the case of the organic compound it is 12-aminolauric acid and/or 12-aminolauric acid methyl ester, which is prepared in the aqueous phase by a strain of E. coli recombinant by stepwise oxidation of the terminal carbon atom of lauric acid methyl ester as published in DE10200710060705 and the hydrophobic phase comprises 25 to 75% oleic acid as liquid cation exchanger dissolved in lauric acid methyl ester as substrate of the reaction.
    [00081] The study of the present invention can be cited not only with the use of exact amino acid or nucleic acid sequences, but also with the use of variants of such macromolecules, which can be obtained through deletion, addition or substitution of one or more of one amino acid or nucleic acid. In the preferred embodiment, the term "variant" means a nucleic acid sequence or amino acid sequence, hereafter used synonymously and interchangeable with the term "homologon", as used herein, another nucleic acid or amino acid sequence. amino acids which, with respect to the respective wild-type nucleic acid sequence or the original amino acid sequence has a homology, used herein synonymously with identity, of 70, 75, 80, 85, 90, 92, 94, 96, 98, 99% or more per cent, preferably other different amino acids forming the catalytically active center or amino acids essential to the structure or folding are deleted or replaced or the latter are merely conservatively substituted, for example, a glutamate instead of an aspartate or a leucine instead of a valine. Prior art describes algorithms that can be used to calculate the extent of homology of two sequences, eg Arthur Lesk (2008), Introduction to bioinformatics, 3rd edition. In another preferred embodiment of the present invention, the variant of an amino acid or nucleic acid sequence, preferably in addition to the sequence homology mentioned above, exhibits essentially the same enzymatic activity as the wild-type molecule or the parent molecule. For example, a variant of an enzymatically active polypeptide as a protease has the same or essentially the same proteolytic activity as the polypeptide enzyme, that is, the ability to catalyze the hydrolysis of a peptide bond. In a particular embodiment, the term "essentially the same enzymatic activity" means an activity with respect to wild-type peptide substrates, which is clearly above the basic activity and/or is distinguished by less than 3, more preferably 2 , even more preferably in an order of magnitude of the KM and/or kcat values, which presents the wild-type polypeptide with respect to the same substrates. In another preferred embodiment, the term "variant" of a nucleic acid or amino acid sequence comprises at least a part/or fragment of the nucleic acid or amino acid sequence. In another preferred embodiment, the term "active part", as used herein, means an amino acid sequence or a nucleic acid sequence, which is shorter in length than the full length of the amino acid sequence or encodes a shorter length. than the total amino acid sequence, where the amino acid sequence or the encoded amino acid sequence of shorter length than the wild-type amino acid sequence has essentially the same enzymatic activity as the wild-type polypeptide or a variant thereof, by example as alcohol dehydrogenase, monooxygenase or transaminase. In a particular embodiment, the term "variant" of a nucleic acid comprises a nucleic acid whose complementary strand, preferably under stringent conditions, binds to wild-type nucleic acid. The stringency of the hybridization reaction can be easily determined by the artisan and generally depends on probe length, temperatures during growth and salt concentration. In general, longer probes need higher temperatures to hybridize, whereas shorter samples live with lower temperatures. Whether hybridization takes place generally depends on the ability of the denatured DNA to anneal into complementary strands, which are present in its medium and, in fact, below the melting temperature. The stringency of the hybridization reaction and its conditions are described in detail in Ausubel et al. 1995. In a preferred embodiment, the term "variant" of a nucleic acid comprises, as used herein, an arbitrary nucleic acid sequence, which encodes the same amino acid sequence as the original nucleic acid or a variant of that amino acid sequence within the scope of the degeneration of the genetic code.
    [00082] Suitable polypeptides, which can be used for the preparation of organic compounds of the Formula (I), in particular, alkanemonooxygenases, AlkL, transaminases, aldehyde dehydrogenases and alanine dehydrogenases, are described in the state of the art, by example, in DE10200710060705, EP11004029 or PCT/EP2011/053834.
    [00083] In the most preferred embodiment, the alkanomonooxygenase is an alkanemonooxygenase of the AlkB type. AlkB represents an oxidoreductase of the AlkBGT system of Pseudomonas putida, which is known for its hydroxylase activity. It depends on two other polypeptides, AlkG and AlkT. AlkT is characterized as FAD-dependent rubredoxin reductase, which transmits electrons from NADH to AlkG. AlkG is a rubredoxin, an iron-containing redox protein that acts as a direct electron donor to AlkB. In a preferred embodiment, by the term "alkB-type alkanomonooxygenase" as used herein is meant a membrane-bound alkanomonooxidase. In another preferred embodiment, by the same term "alkB-type alkanomonooxygenase" is meant a polypeptide having a preferably increasing sequence homology of at least 75, 80, 85, 90, 92, 94, 96, 98 or 99% against the AlkB sequence of Pseudomonas putida Gpo1 (database code: CAB54050.1). In another preferred embodiment, by the term is meant a cytochrome-independent monooxygenase. In another preferred embodiment, by the term "alkB-type alkanomonooxygenase" is meant a cytochrome-independent monooxygenase, which uses at least one rubredoxin or homologon as electron donor. In a particularly preferred embodiment, by the term is meant a membrane-bound cytochrome-independent alkanemonooxygenase, preferably increasing from at least 60, 70, 80, 80, 85, 90, 92, 94, 96, 98 or 99% in relation to the AlkB sequence of Pseudomonas putida Gpo1, which as an electron donor needs at least AlkG (CAB54052.1), but preferably the combination of AlkG with AlkT reductase (CAB54063.1), in the case of alkG and/or alkT is also a homologon of the respective polypeptide. The term "sequence", as used herein, may refer to the amino acid sequence of a polypeptide and/or the nucleic acid sequence which codes for that purpose. In another preferred embodiment, an "alkB-type alkanomonooxygenase" as used herein is a cytochrome-independent oxidoreductase, i.e., an oxidoreductase, which does not comprise cytochrome as a cofactor.
    [00084] The present invention is further illustrated by the following figures and non-restrictive examples, of which other characteristics, embodiments, aspects and advantages of the present invention can be shown.
    [00085] Figure 1 shows a control experiment to confirm that LSME is non-toxic, examined with an E. coli W3110 strain and compared to potassium phosphate buffer (Kpi) as a negative control.
    [00086] Figure 2 shows the life capacity of the E. coli W3110 strain in the form of the number of cfus that the strain can form in the absence of a liquid cation exchanger and in the presence of several liquid cation exchangers after 0 h , 4 h and 24 h.
    [00087] Figure 3 shows the effect of using a liquid cation exchanger on toxicity based on changing the number of viable cells of an E. coli-W3110 strain in the presence of 0.2% ALSME, DEHPA adjusted with ammonia ("D2EHPNH3 2%") or with a mixture of DEHPA/LSME (2%/98%) ("D/L") in the presence of ALSME 0.2%.
    [00088] Figure 4 shows the effect of several liquid cation exchangers on the OTR of the E. coli strain producing aminolauric acid methyl ester. The experiment was carried out as described in example 4.
    [00089] Figure 5 shows the influence of several liquid cation exchangers on the yield of aminolauric acid methyl ester, which produces a strain of E. coli with genetically appropriate modification. The experiment was carried out as described in example 4.Example 1: Research for the toxicity of the LSME solvent, which is used in compositions with liquid cation exchangers.
    [00090] With this test it was shown the relatively low toxicity of LDME with respect to biotechnologically relevant microorganisms, which makes the LSME in a suitable organic solvent for the process according to the invention. an LB plate (10 g/L peptone from casein, 5 g/L yeast extract, 10 g/L NaCl) was streaked with E. coli BW3110 and incubated for 24 hours. In the evening of the following day, a preculture from this previously scratched plate was inoculated. This preculture had a volume of 50 ml of LB medium and was incubated overnight for about 16 hours. The following day, the preculture was reinculated with an OD600 of 0.2 in 200 ml of M9 medium (Na2HPO4 6.79 g/L; KH2PO4 3.0 g/L; NaCl 0.5 g/L; NH4Cl 1g /L; 1 mL/L of trace element solution, pH 7.4. Trace element solution: 37% HCl (=455.8 g/L) 36.50 g/L; MnCl2*7H2O 1.91 g/L; ZnSO4*7H2O 1.87 g/L; Na-EDTA*2H2O (Titriplex III) 0.84 g/L; H3BO3 0.30 g/L; Na2MoO4*2H2O 0.25 g/L; CaCl2*2H2O 4.70 g/L; FeSO4*7H2O 17.80 g/L; CuCl2*2H2O 0.15 g/L) with 3% glucose and incubated for about 20 hours. After incubation of the main culture, cells were harvested, centrifuged at 5258 g and 4°C for 10 minutes and resuspended with an OD600 of 30 in 10 ml of 50 mM Kpi buffer at pH 7.4 (or 25 mM hepes buffer pH 7.4, if cfu determinations were carried out with ALSME). The two buffer solutions used contained 5% glucose. Then, the bacterial suspension was transferred to the shake flasks and mixed with the respective substance solutions. After the mixture is carried out by rotating the flask, 100 μl of the suspension is removed by pipetting and filled into 900 μl of previously introduced sterile saline. These corresponded to the sampling at time t0. Then, the incubation of the preparations was carried out at 250 rotations per minute and 30oC. CFU were determined over a 22 hour period. Samplings were initially carried out at times t0, t3, t6 and t22. In some preparations, another sampling time t1.5 was added and, in addition, another series of additional dilutions, to minimize deviations.
    [00091] The OD600 was 60. The cells were resuspended in 10 ml of Kpi buffer and then mixed in the flask with 5 ml of 98% LSME. A dilution step was performed for each preparation and then inoculated into plates. The number of cfu/ml remained constant over the 6 hour period. After 22 hours, a percentage reduction in the number of viable cells of merely 30.3% was recorded.Example 2: Comparative assays for the toxicity of various liquid cation exchangers towards biotechnologically relevant microorganisms
    [00092] This example shows the low toxicity of unbranched fatty acids compared to other liquid cation exchangers such as DEHPA, as well as branched and unbranched saturated fatty acids.
    [00093] Initially, a preculture comprising 20 ml of LB medium was inoculated in a 100 ml Erlenmeyer flask with a cryoculture of the respective strain. The culture was grown overnight at 37oC and shaken with 200 rotations per minute and used the following day to inoculate the same main culture at an OD of 0.2. Next, the main culture (30 ml each of L(B) medium was still grown under the same conditions. With an OD of 0.4 to 0.5, the main culture was overlaid respectively with the same volumes (30 ml) of solvent and then further cultivated.
    [00094] To determine the number of cfu (colony-forming units or colony-forming units) 0.1 m of samples were removed in the following tests and these were diluted in a sterile 0.9% NaCl solution. Appropriate dilution steps were inoculated onto LB Agar plates. After incubation at 34oC overnight, the formed colonies were counted and the cfus were determined. Assay 1: Comparison of the toxicity between DE2HPA and a saturated fatty acid as a liquid cation exchanger
    [00095] 50% of DEHPA or lauric acid (15%), respectively dissolved in LSME and loaded equimolarly or 25% by mol with ALSME, were brought into contact as liquid cation exchangers with a strain of E. coli BL21 ( DE3) and the influence of these two compounds was investigated for the ability of the strain to form colonies, expressed in cfus. In pre-tests it was possible to show that the lauric acid methyl ester - which due to lack of charge cannot act as a liquid cation exchanger - is well tolerated by the strains used.

    [00096] It appears that the two liquid cation exchangers clearly decrease the number of cfus when lauric acid is used, as opposed to DEHPA, but there are still some viable cells and saturated fatty acid and, therefore, it is given preference to saturated fatty acid as liquid cation exchanger. Assay 2: Comparison of toxicity between branched saturated fatty acids and different amounts of oleic acid as liquid cation exchanger
    [00097] Here, two different concentrations of oleic acid were used and the volume is adjusted by adding the respective amount of LSME (lauric acid methyl ester).

    [00098] It is found that the number of viable cells when oleic acid unsaturated fatty acid is used together with LSME is consistently markedly higher than when branched saturated fatty acids are used.Trial 3: Comparison of toxicity between unbranched saturated fatty acids and unsaturated fatty acids as liquid cation exchanger
    [00099] In this case, various amounts of an unsaturated fatty acid were compared with an unsaturated fatty acid with respect to its toxicity when used as a liquid cation exchanger. Based on the lower solubility of the unsaturated fatty acid lauric acid, this was used in a smaller amount. The volumes of the various liquid cation exchangers were compared with LSME. The number of cfus was determined at the beginning, after 4.5 hours and after 24 hours.
    [000100] As can be seen in Fig.2, the addition of the saturated fatty acid as a liquid cation exchanger even at a lower concentration than the unsaturated fatty acid causes a reduction in the cfus, whereas in the case of the unsaturated fatty acid , there is an increase in cfus.
    [000101] Altogether, there is a decrease in toxicity in the various liquid cation exchangers searched in the following order: DEHPA > saturated fatty acids > unsaturated fatty acids.Example 3: Decreased toxicity of a positively charged organic compound through contact with a liquid cation exchanger
    [000102] This test shows that through the presence of a liquid cation exchanger, the toxic effect of a positively charged organic compound in an aqueous phase, which is fermentation broth, can be reduced, in which this compound is extracted in the organic phase.
    [000103] The basic experimental procedure corresponded to that of example 1.
    [000104] Since 0.2% ALSME, dissolved in aqueous systems, acted as a bactericide, this assay was performed in combination with D2EHPNH3/LSME 2/98% again in the shake flask, in this case, D2EHPNH3 means D2EHPA quantitatively loaded with ammonium. Through the use of a liquid ion exchanger, the passage of ALSME to the organic phase is improved, so that this concentration in the aqueous phase, in which the cells are also found, is reduced. To reduce the toxic effect conditioned by D2EHPA, reduced concentrations of 2% of D2EHPNH3 were used.
    [000105] Initially, the bacteria were resuspended in 5 mL (corresponds to half the volume of the buffer). A further 5 ml of buffer was optionally mixed with 0.4% ALSME and then optionally centrifuged with 5 ml of D2EHPNH3/LSME 2/98% for 1 minute at 300 revolutions per minute. This solution was added to the suspension of bacteria previously introduced into the shake flask and mixed. Afterwards, the first sampling was carried out.
    [000106] The solution had a foamy consistency at the beginning of the tests, which, however, in the second sampling, had disappeared in both tests. The abbreviation "D/L" was used for D2EHPNH3 (D2EHPA)/LSME 2/98% loaded with ammonia. Between t0 and t1.5 hour samples taken, the number of cfu/mL increased by 34.3%. From sampling (t1.5) to the last sampling (t22) the number of cfu/mL decreased by 54.9%. Compared to the preparation with D2EHPNH3/LSME 2/98% without addition of 0.2% ALSME, the number of cells capable of propagation after 22 hours was 4.5 times higher and with 3.4% not significantly lower than the mean value of the control preparations in hepes buffer (see figure 4). Compared to the preparation with 0.2% ALSME in the shake flask, without addition of an organic phase, the number of cfu/ml was 2800 times higher.
    [000107] It is found that the presence of the liquid cation exchanger reduces the toxicity of the positively charged compound, here verified by the number of remaining cfus.Example 4: Comparative tests for the toxicity of several liquid cation exchangers in relation to an acid w- aminolauric (ALS) and the methyl ester producing microorganism (ALSME)
    [000108] The biotransformation of lauric acid methyl ester to aminolauric acid methyl ester was tested in the 8 times larger parallel fermentation system of DasGip with several ion exchangers.
    [000109] For the fermentation 1 liter reactors were used. The pH probes were calibrated by means of a two-point calibration with measuring solutions of pH 4.0 and pH 7.0. The reactors were filled with 300 mL of water and autoclaved at 121oC for 20 minutes to ensure sterility. Then, pO2 probes were polarized overnight (for at least 6 hours). The next morning, the water was removed under the clean bench and replaced with a high cell density medium with 50 mg/L of kanamycin and 34 mg/L of chloramphenicol. Next, the pO2 probes were calibrated with a one-point calibrator (stirrer: 600 revolutions per minute/gasification: 10 sl/h of air) and the feeding routes, corrective agent and induction medium were purified through cleaning in the clean-in-place. For this, the hoses were rinsed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and, finally, filled with the respective media.
    [000110] The E. coli strain BL21 (DE3) T1r pBT10 pACYC:Duet[TAcv] producing ALS and ALSME was initially cultivated from cryoculture in LB medium (25 ml in a 100 ml Erlenmeyer) with 50 mg/ L of kanamycin and 34 mg/L of chloramphenicol overnight at 37oC and 200 rotations per minute, for about 18 hours. Then, 2 mL each of high cell density medium (15 g/L glucose (30 mL/L of 500 g/L of a standard solution autoclaved separately with 1% MgSO4*7H2O and 2.2% NH4Cl), (NH4 )2SO4 1.76 g/L, K2HPO4 19.08 g/L, KH2PO4 12.5 g/L, yeast extract 6.66 g/L, trisodium citrate dihydrate 11.2 g, iron-citrate solution ammonium 17 mL/L of a separately autoclaved 1% standard solution, 5 mL/L of a separately autoclaved standard solution of trace elements (HCl 37%) 26.50 g/L, MnCl2*4H2O 1.91 g/L, ZnSO4*7H2O 1.87 g/L, ethylenediaminetetraacetic acid dihydrate 0.84 g/L, H3BO3 0.30 g/L. Na2MoO4*2H2O 0.25 g/L, CaCl2*2H2O 4.70 g/L , FeSO4*7H2O 17.80 g/L, CuCl2*2H2O 0.15 g/L)) (3 times 25 mL each in a 100 mL Erlenmeyer flask) was reinculated with 50 mg/L of kanamycin and 34 mg/L of chloramphenicol and incubated at 37oC/200 rotations per minute for another 6 hours.
    [000111] The 3 cultures were combined in a shake flask and the optical density was determined to be 7.2. To inoculate the reactors with an optical density of 0.1, 4.2 ml each were applied in a 5 ml syringe and the reactors were inoculated through cannulas through a septum.
    [000112] The following standard program was used:

    [000113] The experiment carried out can be broken down into two phases, the culture, in which the cells must obtain a certain optical density and the subsequent biotransformation, in which the expression of the gene necessary for the biotechnological process for the production of ALSME was induced. The pH values were regulated, on the one hand, with ammonia (12.5%) to pH 6.8. During cultivation and biotransformation, dissolved oxygen (DO) in the culture was regulated by the number of rotations of the stirrer and a 30% gassing rate. Fermentation was carried out as a feed mixture, and the start of feeding, 5 g/L h of glucose feed (500 g/L of glucose with 1% MgSO4*7H2O and 2.2% NH4Cl) was triggered by of a peak of DO. With the beginning of feeding, the temperature from 37oC was also reduced to 30oC. Transaminase expression was induced 2 hours after the start of feeding by automatic addition of IPTG (1 mM). The induction of the alk gene was performed by manual addition of DCPK (0.025%) 10 hours after the start of feeding. Before starting the biotransformation, the optical density of the culture broths was determined.
    [000114] The start of the biotransformation phase was performed 14 hours after the start of feeding. For this purpose, 150 ml of a mixture of lauric acid methyl ester and the corresponding ion exchanger (10%) were added as a mixture to the fermentation broth. As ion exchangers, di-(2-ethylhexyl) phosphoric acid (DEHPA), lauric acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid and a mixture of free fatty acids from the saponification of thistle oil were used. spherical flowers. By making an amino group donor available for transaminase, 10.7 ml of an alanine solution (125 g/L) were added to the fermentation broth at the same time with the addition of the organic phase. For sampling, 2 mL of the fermentation broth was removed from the boiler and a part of it, 1/20, was diluted in an acetone-HCl mixture (c(HCl) = 0.1 mol/L) and extracted. Samples were taken from all 8 reactors at 1.25 h, 3 h, 5 h, 20 h, 22 h and 25 h after the start of biotransformation. The conversion rates for oxygen (OTR = oxygen transfer rate) and carbon (CTR = carbon transfer rate) were determined during fermentation through the analysis of residual gases in DasGip systems. Fermentation was completed 22 hours after the start of biotransformation.
    [000115] The quantification of ALS, ALSME, DDS, DDSME, LS, LSME, HLS, HLSME, OLS and OLSME in fermentation samples was performed using LC-ESI/MS2 based on an external calibration for all analytes and with the use of the internal standard aminoundecanoic acid (AUD).
    [000116] In this case, the following devices were used: • HPLC device 1260 (Agilent; Boblingen) with autosampler (G1367E), binary pump (G1312(B) and column oven (G1316A) • TripelQuad 6410 mass spectrometer (Agilent ;Boblingen) with ESI source• HPLC column: Kinetex C18, 100 x 2.1 mm, particle size: 2.6 µm, pore size 100 (Phenomenex;Aschaffenburg)• precolumn: KrudKatcher Ultra HPLC filter in line; 0.5 μm filter depth and 0.004 mm internal diameter (Phenomenex; Aschaffenburg).
    [000117] Samples were prepared, in which 1900 μl of solvent (mixture of acetone/HCl 0.1 N = 1:1) and 100 μl of sample were pipetted into a 2 mL reaction vessel. The mixture was centrifuged for about 10 seconds and then it was centrifuged at about 13,000 revolutions per minute for 5 minutes. The clear supernatant was removed with a pipette and analyzed after its dilution with diluent (80%) ACN, 20% bidistilled H2O + 0.1% formic acid). For every 900 μl of sample, 100 μl of ISTD was added by pipetting (10 μl with a sample volume of 90 μl).
    [000118] HPLC separation was performed with the column or pre-column mentioned above. The injected volume imported in 0.7 μl, the column temperature in 50oC, the flow rate in 0.6 mL/min. The mobile phase consisted of eluent A (0.1% aqueous formic acid) and eluent B (acetonitrile with 0.1% formic acid). The following gradient profile was used:

    [000119] ESI-MS2 analysis was performed in positive mode with the following ESI source parameters: • gas temperature 280oC • gas flow 11 l/min • nebulization pressure 50 psi • capillary voltage 4000 V
    [000120] The detection and quantification of individual compounds was performed with the following parameters, in each case using a product ion as a qualifier and one as a quantifier:

    Results:
    [000121] If DEHPA, as described in the prior art, is used as a liquid cation exchanger, then immediately after adding the compound to the culture, a breakdown of the OTR occurs. The curve soon drops to 0, which indicates that in the culture there are no longer any metabolically active cells. DEHPA therefore acts as highly toxic on cells.
    [000122] If, instead of DEHPA, lauric acid is used as a liquid cation exchanger, then, in fact, there is also a rupture of the OTR, however, this is not as strong and over the next 22 hours, the cells recompose and show an increasing activity of metabolism. Therefore, lauric acid is remarkably less toxic than DEHPA.
    [000123] Even more evident results can be observed when saturated fatty acids with longer carbon chains are used. Using palmitic acid and stearic acid, the OTR curve is markedly safer than using lauric acid or even DEHPA. From this it can be concluded that these fatty acids act in a distinctly less toxic way.
    [000124] The use of unsaturated fatty acids, such as palmitoleic acid, saponified spherical flower thistle oil (contains mainly linolic acid) and oleic acid surprisingly lead to even evidently better results. These fatty acids surprisingly show even lower toxicity than saturated fatty acids.Literary References:J. Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, Vol. 2 of Wiley Series in Solution Chemistry, John Wiley & Sons, Chichester, 1997 Asano, Y., Fukuta, y., Yoshida, Y., and Komeda, H. (2008): The Screening, Characterization, and Use of w-Laurolactam Hydrolase: A New Enzymatic Synthesis of 12-Aminolauric Acid, Biosc. Biotechn. Biochem., 72 (8), 2141-2150DE10200710060705 (2007): Recombinant cells producing w-aminocarboxylic acids or their lactamsF. M. Ausubel (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc.A. M. Lesk (2008), Introduction to Bioinformatics, 3rd edition
    权利要求:
    Claims (12)
    [0001]
    1. Process for removing an organic compound from an aqueous solution, characterized in that it comprises the steps of: (a) providing the aqueous solution containing the organic compound and a hydrophobic organic solution, which comprises a liquid cation exchanger, the liquid cation exchanger being hydrophobic, (b) contacting the aqueous solution with the organic solution, and (c) separating the organic solution from the aqueous solution, the organic compound being a compound of the Formula (I)NH3+-A -COOR1 (I), in which R1 is hydrogen, methyl, ethyl or a negative charge, and A is an unsubstituted, straight-chain alkylene group with at least three, preferably with at least eight carbon atoms, and the exchanger of liquid cation is an unsaturated fatty acid.
    [0002]
    2. Process according to claim 1, characterized in that the temperature in step (b) is from 28 to 70oC, preferably 30 to 37oC.
    [0003]
    3. Process according to claim 1 or 2, characterized in that the pH value in step (b) is from 6 to 8, preferably 6.2 to 7.2.
    [0004]
    4. Process according to any one of claims 1 to 3, characterized in that the molar ratio of the liquid cation exchanger to the organic compound is at least 1.
    [0005]
    5. Process according to any one of claims 1 to 4, characterized in that the volumetric ratio of organic solution to aqueous solution is from 1:10 to 10:1.
    [0006]
    6. Process according to any one of claims 1 to 5, characterized in that the liquid cation exchanger is a fatty acid having more than 12, preferably having from 14 to 22, even more preferably having 16 to 18 carbon atoms.
    [0007]
    7. Process according to any one of claims 1 to 6, characterized in that the liquid cation exchanger is oleic acid or erucic acid.
    [0008]
    8. Process according to any one of claims 1 to 7, characterized in that the aqueous solution comprises a cell.
    [0009]
    9. Process according to claim 8, characterized in that the cell is a bacterial cell, and the cell most preferably includes a recombinant alkanemonooxygenase, a recombinant transaminase, and preferably, in addition, at least one enzyme from the group comprising an alcohol dehydrogenase, an alanine dehydrogenase and the AlkL gene product.
    [0010]
    10. Process according to any one of claims 1 to 9, characterized in that the organic solution additionally contains at least one organic solvent, preferably a fatty acid and/or a fatty acid ester.
    [0011]
    11. Process according to claim 10, characterized in that the organic solution comprises: as a liquid cation exchanger, 20 to 80% by volume, preferably 25 to 75% by volume, of oleic acid, and as a solvent, lauric acid methyl ester, and the organic compound is methyl 12-aminolaurate, and a bacterial cell is present in the aqueous solution, including a recombinant alkanemonooxygenase, a recombinant transaminase, and preferably, in addition, at least one enzyme of the group , which comprises an alcohol dehydrogenase, an alanine dehydrogenase and the gene product AlkL.
    [0012]
    12. Reaction mixture, characterized in that it comprises: an aqueous solution, obtained by a process as defined in any one of claims 1 to 11, and a hydrophobic organic solution, wherein the hydrophobic organic solution comprises a fatty acid, plus preferably a fatty acid having more than 12 carbon atoms, even more preferably an unsaturated fatty acid as a liquid cation exchanger.
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    同族专利:
    公开号 | 公开日
    SG192825A1|2013-09-30|
    EP2489432A3|2012-12-19|
    US20140039210A1|2014-02-06|
    MX344165B|2016-12-07|
    EP2489432B1|2017-08-23|
    JP5936627B2|2016-06-22|
    KR101867119B1|2018-07-17|
    UA112980C2|2016-11-25|
    CN103459032B|2017-02-15|
    MX2013009463A|2013-09-06|
    US10071951B2|2018-09-11|
    CA2826414C|2019-04-23|
    WO2012110124A1|2012-08-23|
    SG10201913938SA|2020-03-30|
    CN103459032A|2013-12-18|
    AU2011359126A1|2013-09-05|
    CA2826414A1|2012-08-23|
    US9315443B2|2016-04-19|
    ES2642237T3|2017-11-15|
    RU2592287C2|2016-07-20|
    SG192823A1|2013-09-30|
    KR20140016285A|2014-02-07|
    WO2012110125A1|2012-08-23|
    WO2012110126A1|2012-08-23|
    US20140054224A1|2014-02-27|
    BR112013020912A2|2016-10-04|
    MY164856A|2018-01-30|
    CN103459033B|2016-08-10|
    EP2489432A2|2012-08-22|
    RU2013141946A|2015-03-27|
    JP2014511263A|2014-05-15|
    CN103459033A|2013-12-18|
    AU2011359126B2|2016-06-09|
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    法律状态:
    2018-11-13| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: B01J 39/04 , B01J 39/16 , B01D 11/04 , C07C 227/40 , C13B 20/14 , C02F 1/42 Ipc: C02F 1/42 (1980.01), B01J 39/16 (1980.01), B01D 11 |
    2018-12-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-03-24| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-05-12| B25D| Requested change of name of applicant approved|Owner name: EVONIK OPERATIONS GMBH (DE) |
    2020-10-27| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|
    2021-03-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-06-01| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP11154707.1|2011-02-16|
    EP11154707|2011-02-16|
    PCT/EP2011/071491|WO2012110124A1|2011-02-16|2011-12-01|Liquid cation exchanger|
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