WO2012007023A1 - Procédé de recyclage d'un biocatalyseur dans un système de solvants à plusieurs constituants à température commandée - Google Patents

Procédé de recyclage d'un biocatalyseur dans un système de solvants à plusieurs constituants à température commandée Download PDF

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WO2012007023A1
WO2012007023A1 PCT/EP2010/004357 EP2010004357W WO2012007023A1 WO 2012007023 A1 WO2012007023 A1 WO 2012007023A1 EP 2010004357 W EP2010004357 W EP 2010004357W WO 2012007023 A1 WO2012007023 A1 WO 2012007023A1
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general formula
component
reaction medium
reaction
polysubstituted
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PCT/EP2010/004357
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German (de)
English (en)
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Arno Behr
Leif Johnen
Bastian Daniel
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Technische Universität Dortmund
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Priority to PCT/EP2010/004357 priority Critical patent/WO2012007023A1/fr
Publication of WO2012007023A1 publication Critical patent/WO2012007023A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6418—Fatty acids by hydrolysis of fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to a process for reacting a starting material to form a product in the presence of a biocatalyst and optionally an excipient in a one-phase reaction medium comprising at least two components of different polarity, followed by separation of the reaction products and the biocatalyst in one Temperature T 2 at which the reaction medium is at least biphasic.
  • biocatalysts such as enzymes in the process industry is becoming increasingly important. To date, however, comparatively few processes are known in which the usually very expensive biocatalyst can be separated cost-effectively and reused without or with only slight losses of activity. However, the most complete and therefore economically sensible separation of the biocatalyst after a successful reaction and the associated reusability of the catalyst are essential for the success of a process.
  • biocatalysts such as enzymes in reusable form, for example, their immobilization via adsorption to suitable support materials or the inclusion in aqueous gels.
  • Disadvantages of this approach are the leaching of the enzymes and often also a significant reduction in activity.
  • the use of relatively large amounts of non-catalytic material adversely affects the required reaction volume and reaction times.
  • the inclusion of an enzyme in aqueous gels also requires the complex synthesis of a polymer in the presence of the enzyme.
  • enzymes can be separated from the reaction mixture by special membranes which selectively allow only the products to pass.
  • suitable membranes have to be developed very expensive, have to be very selective, are often very expensive and clog easily in continuous process control.
  • Another method is the use of liquid-liquid two-phase systems in which aqueous medium and a non-miscible organic medium are used.
  • aqueous medium and a non-miscible organic medium are used.
  • Another possibility for realizing enzymatic reactions of hydrophobic substances is the use of emulsions, which first have to be produced by great stirring effort and using a generally expensive surfactant. Although the mass transfer is facilitated here, but the long and fuzzy phase separation of the aqueous catalyst phase and the organic product phase usually prevent a technical use.
  • An object of the invention was to provide new methods that do not have the disadvantages over the prior art, or at least in reduced form. In particular, these methods should allow the simple implementation of biocatalytic reactions and a simple and cost-effective reuse of the biocatalyst used.
  • the present invention relates to a method for biocatalyzed reaction comprising at least the steps
  • step (b) cooling or heating the reaction medium (R) to a temperature T 2 at which the reaction medium (R) is biphasic, wherein the biocatalyst (B) and optionally employed (r) excipient (s) substantially in one phase ( P1) of the biphasic reaction medium (R) and the product (P), optionally unreacted starting material (E) and optionally used (r) excipient (s) substantially in the other phase (P2) of the biphasic reaction medium (R) .
  • step (c) separating the two phases of the reaction medium (R) obtained in step (b) from one another and
  • step (D) workup and / or purification of the recovered in step (c) phase (P1) and reuse of the biocatalyst (B) or direct reuse of the phase (P1) and the contained biocatalyst (B) without prior workup and / or purification ,
  • the process according to the invention for biocatalyzed conversion is characterized in that the reaction medium (R) consists of two or more components and, depending on the temperature, is in the form of a single phase or two phases. During the catalyzed reaction, the system is single-phase. To terminate the reaction or after the reaction, the reaction medium can be converted into a two-phase state.
  • the components of the reaction medium are in particular to be chosen such that the biocatalyst (B) is used in one phase (P1) of the biphasic reaction medium (R) and the product (P), optionally unreacted starting material (E) (R) excipient (s) and optionally formed by-product (s) substantially in the other phase (P2) of the biphasic reaction medium (R) are present.
  • the biocatalysts according to the invention can easily be separated off from the products (P) and optionally further substances and used again.
  • substantially in the sense of the present invention means that preferably at least 75% by weight, more preferably at least 80% by weight, particularly preferably at least 85% by weight, very preferably at least 90% by weight and most preferably at least 95% by weight of the biocatalyst is in phase (P1).
  • the other substances ie the product (P), optionally unreacted starting material (E), optionally used auxiliaries) and optionally formed by-product (s) are in particular 50% by weight, preferably at least 75 % By weight, more preferably at least 80% by weight, more preferably at least 85% by weight, most preferably at least 90% by weight and most preferably at least 95% by weight in the other phase (P2) of Reaction medium before.
  • a separation of the biocatalyst from the other substances can thus be achieved in a simple and therefore also cost-effective manner by separating the phases (P1) and (P2).
  • the catalyst phase can then be used directly, that is to say without prior work-up and / or purification, in a further cycle according to the process of the invention.
  • the phase (P1) contains further substances after its separation which make it advantageous to work up and / or purify this phase, this can be carried out by customary methods known to the person skilled in the art.
  • reaction medium (R) in the biocatalytic reaction process of the invention basically all solvent mixtures are considered, which pass by a change in temperature from a single-phase state to a two-phase state, ie their miscibility shows a temperature-dependent behavior in the temperature range between and T 2 .
  • Such systems are also known as temperature controlled multi-component solvent (TML) systems.
  • TTL temperature controlled multi-component solvent
  • the transition from a single-phase state to a two-phase state according to process step (b) is achieved by cooling, ie T ⁇ T ⁇ but those skilled in the art are also known systems in which a single-phase reaction medium is generated by cooling, ie Ti ⁇ T 2 . Both systems are encompassed according to the invention, but preferably, according to process step b), the reaction medium (R) is cooled to a temperature T 2 at which the reaction medium (R) is biphasic.
  • the change in phase behavior i. the transition from the single-phase system to the two-phase system, between -50 ° C to 200 ° C, preferably between 0 ° C to 100 ° C, more preferably between 0 ° C to 85 ° C and most preferably between 20 ° C and 70 ° C instead.
  • all indicated temperatures are preferably based on normal pressure.
  • reaction medium usually one reaction medium (system) of two or more components is used, wherein the use of a reaction medium of three components is preferred. All temperatures given in the present application are preferably based on atmospheric pressure.
  • the component (L1) is nonpolar.
  • nonpolar component (L1) one or more non-polar solvents may be present, such as for example
  • long-chain hydrocarbons preferably C 5 -C 8 -aliphatic hydrocarbons, more preferably C 6 -C 12 -aliphatic hydrocarbons, in each case saturated or unsaturated, branched or unbranched, unsubstituted or monosubstituted or polysubstituted (in particular n-pentane, n-hexane, n-heptane, n-octane, N-nonane, octene, dichloromethane, trichloromethane);
  • cycloaliphatic hydrocarbons preferably C 6 -C 2 -cycloaliphatic hydrocarbons, in each case saturated or unsaturated, unsubstituted or monosubstituted or polysubstituted, (in particular cyclohexane);
  • aromatic hydrocarbons preferably C 6 -C 4 aromatic hydrocarbons, more preferably C 6 -C 0 aromatic hydrocarbons, unsubstituted or mono- or polysubstituted (particularly benzene, toluene, chlorobenzene);
  • Alcohols of the general formula R 1 -OH (in particular cyclohexanol, hexanol, phenol)
  • Ethers of the general formula R 1 -O-R 1 (in particular dipentyl ether, diphenyl ether)
  • R a in each occurrence independently of one another Ci 0 -C 14 aliphatic hydrocarbon, in each case saturated or unsaturated, branched or unbranched, unsubstituted or mono- or polysubstituted; means;
  • R b each occurrence independently of one another C 7 -C 4 -aliphatic hydrocarbon, in each case saturated or unsaturated, branched or unbranched, unsubstituted or monosubstituted or polysubstituted; means;
  • R 1c at each occurrence is independently a C 10 -C 5 aliphatic hydrocarbon, in each case saturated or unsaturated, branched or unbranched, unsubstituted or mono- or polysubstituted.
  • At least one component (L2a) and / or at least one component (L2b) is present as component (L2).
  • the component (L2a) is polar.
  • one or more polar solvents may be present, such as for example
  • polyhydric alcohols in particular ethanediol, glycerol, propanediol, sorbitol
  • ionic liquids especially 1-butyl-3-methylimidazolium hexafluorophosphate, 1-ethyl-3-methylimidazolium tetrafluoroborate).
  • polar solvents such as a combination of water and ionic liquid.
  • the component (L2b) is of medium polarity.
  • medium-polar component (L2a) one or more medium-polar solvents may be present, such as for example
  • monohydric alcohols of the general formula R 2 -OH especially methanol, ethanol, isopropyl alcohol
  • cyclic carbonates having up to 7 C atoms in the ring (especially ethylene carbonate, propylene carbonate, butylene carbonate, glycerin carbonate and its esters);
  • Lactones having up to 7 C atoms in particular propiolactone, butyrolactone
  • Lactams with up to 6 carbon atoms especially gamma-butyrolactam
  • Heteroaromatic compounds having up to 5 carbon atoms and 1, 2 or 3 heteroatoms selected from the group consisting of N, O and S, preferably 5- or 6-membered, (in particular pyridine, diazine, oxazine, azole, imidazole, pyrazole , Furan, oxazole); in which
  • R 2 each occurrence independently of one another d-Cs-aliphatic hydrocarbon, in each case saturated or unsaturated, branched or unbranched, unsubstituted or mono- or polysubstituted; means
  • R 3 each occurrence independently of one another d-Cr-aliphatic hydrocarbon, in each case saturated or unsaturated, branched or unbranched, unsubstituted or mono- or polysubstituted; means, and
  • R 4 independently of one another independently of one another represent a C 1 -C 2 aliphatic hydrocarbon, in each case saturated or unsaturated, branched or unbranched, unsubstituted or monosubstituted or polysubstituted; means.
  • radicals such as, for example, R 1 , R 2 , R 3 and R 4 occur several times in a compound, these can each be selected independently of one another from the list of the meanings indicated.
  • Aliphatic and cycloaliphatic hydrocarbons in the context of the present invention in each case comprise saturated and unsaturated hydrocarbons, ie. Alkanes, alkenes and alkynes or cycloalkanes, cycloalkenes and cycloalkynes.
  • a reaction medium of 3 components is used, namely a non-polar component (L1), a polar component (L2a) and a medium-polar component (L2b).
  • a reaction medium of 2 components is used, namely a non-polar component (L1) and a polar component (L2a) or a medium-polar component (L2b).
  • an educt in particular if it is a non-polar or medium-polar educt, can also function as a component of the reaction medium (L1, L2, L2a, L2b).
  • This variant of the method is particularly advantageous because one or more components of the system can thus be saved.
  • the transesterification of a fat with methanol may be mentioned here.
  • Methanol and fat are the educts, with methanol at the same time being the polar solvent for accepting the catalyst and the fat being the non-polar solvent.
  • a classification of the components or solvents in nonpolar, middle polar and polar can preferably be carried out via the Hansen parameter ( ⁇ 0 ), which was developed via cohesion energy parameters and has been widely used for the characterization of liquids.
  • Cohesion energy parameters are also referred to as Hansen Solubility Parameters (HSP).
  • HSP Hansen Solubility Parameters
  • the Hansen parameter defines the polarity over the cohesive forces that occur in a liquid.
  • the energy of the cohesive forces is divided into three categories: non-polar atomic interaction (dispersive component) (E d );
  • the Hansen parameter ( ⁇ 0 ) is given in the unit MPa 0.5 is temperature-dependent.
  • a reaction medium of 2 components is used, namely a nonpolar component (L1) having a ( ⁇ 0 ) in the range from 10.0 to 22.0 MPa 0.5 and a polar component ( L2a) with one ( ⁇ 0 ) in the range of 22.1 to 50.0 MPa 0.5 .
  • a reaction medium of 3 components is used, namely a nonpolar component (L1) having a ( ⁇ 0 ) in the range from 10.0 to 19.0 MPa 0.5 and a polar component ( L2a) having one ( ⁇ 0 ) in the range of 26.5 to 50.0 MPa 0.5 and a mid-polar component (L2b) of one ( ⁇ 0 ) in the range of 20.0 to 29.9 MPa 0.5 .
  • L1 nonpolar component
  • L2a polar component having one ( ⁇ 0 ) in the range of 26.5 to 50.0 MPa 0.5
  • a mid-polar component (L2b) of one ( ⁇ 0 ) in the range of 20.0 to 29.9 MPa 0.5 a reaction medium of 3 components
  • a biocatalyst for the purposes of the present invention is a polymeric biomolecule or an organism that can catalyze a chemical reaction.
  • the biocatalyst used is at least one enzyme and / or at least one microorganism.
  • the reaction according to the method of the invention it is also possible to use mixtures of two or more enzymes or mixtures of two or more microorganisms or mixtures of at least one enzyme and at least one microorganism. Usually, however, only one enzyme or one microorganism is used at a time.
  • the enzymes can be used in free (isolated) or in immobilized form, with basically all enzymes known to those skilled in the art also including those from extremophilic organisms.
  • the enzymes used according to the invention may be selected from the group consisting of Oxidoreductases, preferably dehydrogenases, hydrogenases, oxidases, oxygenases, reductases and hydroxylases;
  • Transferases preferably phosphotransferases and aminotransferases
  • Hydrolases preferably esterases, lipases, phosphatases and peptidases
  • Lyases preferably decarboxylases, aldolases and synthases
  • Isomerases preferably mutases and racemases
  • Ligases preferably synthetases and carboxylases.
  • lipases may preferably be selected from the group consisting of Candida antarctica lipase B, Candida rugosa lipase, Pseudomonas cepacia lipase, Rhizomucor mihei lipase, Pseudomonas fluorescens lipase, Candida cylindracea lipase, Arthrobacter sp. Lipase and Serratia marescens lipase.
  • biocatalyst may be a whole-cell biocatalyst, including extremophilic organisms.
  • fungi such as Saccharomyces cerevisiae
  • bacteria such as Escherichia coli or archaea are mentioned.
  • auxiliaries (H) can be used.
  • Suitable auxiliaries and / or additives are, for example, buffer substances which are capable of keeping the pH of the reaction medium constant within a certain range after addition of an acid / base or reaction-induced formation of an acid / base. These buffers may be added as a whole buffer system before the reaction or, during the reaction, the corresponding acid / base may be added to the buffer system used.
  • suitable buffer systems include acetic acid / sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, ammonia buffer (NH 3 + H 2 O + NH 4 Cl), tris (hydroxymethyl) -aminomethane (Tris) / hydrochloric acid and triallylamine / hydrochloric acid.
  • lipases as catalysts in the following reactions: kinetic resolutions such as the hydrolysis of chiral carboxylic esters to optically active alcohols and optically active ethers, the enantioselective esterification of chiral alcohols to optically active alcohols and optically active esters or the enantioselective amidation of amines with esters; complete or selective hydrolysis of esters such as lipid cleavage to free fatty acids and glycerol or the 1,3-selective cleavage of a fat to a free fatty acid and a 2-substituted glycerol;
  • esters such as e.g. in the production of biodiesel; as well as the
  • Esterification or partial esterification of alcohols e.g. in the preparation of sugar esters such as sugar acrylates or the production of isopropyl palmitate and isopropyl myristate.
  • reaction medium can be prepared from industrially accessible components (solvents), i.
  • solvents there is an extremely large number of very inexpensive solvents available.
  • phase behavior in particular temärer solvent systems usually from extraction systems is known, so that a time-consuming search for suitable components - such as in the use of emulsions or microemulsions - is not necessary.
  • a scale-up to the production scale is therefore also easily possible.
  • reaction according to the invention proceeds in a single phase, there is no mass transfer limitation, which has an advantageous effect on the yields of the desired product and the reaction times.
  • method according to the invention is basically suitable for any type of reaction and the ease of transfer from one type of reaction to another is also very easily possible.
  • the process according to the invention can be carried out continuously or batchwise.
  • FIG. 1 shows the course of the reaction of para-palmitic acid nitrophenyl ester (pNPP) to para-nitrophenol (pNP) and palmitic acid in the presence of lipase.
  • FIG. 2 shows the course of the first and second conversion of pNPP to pNP in the presence of the same catalyst phase.
  • FIG. 3 represents the yield of pNP after 5 reaction cycles.
  • FIG. 4 shows the course of the reaction of pNPP to pNP in a biphasic
  • the enzyme used as well as the chemicals and solvents used were obtained commercially from the conventional suppliers, namely the lipase Amano Lipase PS from Amano Pharmaceuticals Ltd. (Nagoya, Japan) and para Palmitin Textrenitrophenylester (98% +) and para-Palmitin yarnrenitrophenylester for calibrations (Pestanal ®, analytical standard) from Sigma-Aldrich (St. Louis, MO, USA). All non-aqueous solvents were purchased from Acros Organics (Geel, Belgium). All of these solvents had a purity of at least 99% with the exception of hexanol, which had a purity of 98% +. All other reagents used were also commercially available from the traditional suppliers and each had the highest purity available. Photometric determinations were carried out on a Specord S600 from Analytik Jena AG (Jena, Germany).
  • the product and educt concentrations were determined by means of high performance liquid chromatography (HPLC). These determinations were carried out on a computer controlled Merck-Hitachi HPLC system (VWR International, Darmstadt, Germany) containing the following modules, namely: an interface L-7000, a pump L-7100, a diode array detector L-7450, a Auto sampler L-7200 with a 100 ⁇ sample loop, a solvent degasser L-7612 and a high-pressure gradient mixer. The control of the device as well as the data acquisition and analysis was carried out with the LaChrom software version 3.2.1.
  • the reaction was carried out in a system of bidistilled water, methanol and hexanol.
  • the biocatalyst used was the lipase Amano Lipase PS.
  • the pH of 7 of the system was adjusted by the addition of triallylamine and dilute hydrochloric acid. It has been verified in advance that under these conditions the hydrolysis of the educt para-palmitic acid nitrophenyl ester without the presence of the lipase is negligible.
  • the system was assembled in a 100 ml wide-mouth flask made up of 5.5 g hexanol, 330 ⁇ l triallylamine, 15.5 g methanol and 23.0 g H 2 O.
  • the system was biphasic at room temperature. To adjust the pH, the system was heated to 40 ° C and added dropwise diluted hydrochloric acid to a pH of 7. Subsequently, 1.0 mg lipase in 1000 ⁇ water was added and the reaction started by addition of 15.0 mg para- Palmitinklarenitrophenylester in 500 mg of hexanol. The reaction was carried out at an oil bath temperature of 45 ° C.
  • reaction mixture 250 ⁇ of the reaction mixture were taken at certain times in each case and immediately mixed with the same volume of a mixture of methanol / trifluoroacetic acid (95/5 v / v).
  • methanol served to produce a monophasic solution and trifluoroacetic acid was used to denature the lipase and thus terminate the reaction.
  • the lipase was stirred for 23 hours in the solvent mixture and at the temperature according to Example 1. Subsequently, 15 mg of para-palmitic acid nitrophenyl ester was added to start the reaction. It has been found that the lipase has a high stability under these conditions and leads to practically identical reaction rates.
  • reaction mixture was transferred to a separatory funnel heated to 5 ° C., and the phases were separated. Each appropriately prepared organic phase was combined with the separated aqueous phase containing the lipase and heated to 45 ° C. Subsequently, the reaction was restarted by addition of an appropriate amount of educt. It has been found that the catalyst phase can be used repeatedly without any noticeable loss of activity.
  • the reaction course of the first and second reaction according to the process of the invention was essentially identical and shown in FIG.
  • Example 1 The reaction carried out in Example 1 according to the invention was also carried out in a two-phase system which does not become monophasic at the reaction temperature. That is, the reaction was similar to a classic liquid / liquid two-phase technology. To this was reacted 15 mg of para-palmitic acid nitrophenyl ester, 21.5 g of hexanol and 23.0 g of a 50 mM solution of a buffer of tris (hydroxymethyl) aminomethane and hydrochloric acid (pH 7) at a temperature of 45 ° C. The reaction was started by adding 1 mg of Lipase Amano Lipase PS.
  • the lipase was dissolved in the aqueous phase in this case, while the educt para- Palmitinklarenitrophenylester was present in the organic phase.

Abstract

L'invention concerne un procédé de transformation d'un produit de départ en produit en présence d'un biocatalyseur et éventuellement d'un agent auxiliaire dans un fluide de réaction monophase à une température T1, composé d'au moins deux constituants de polarités différentes, et de séparation consécutive des produits de réaction et du biocatalyseur à une température T2, à laquelle le fluide de réaction est présent sous forme au moins diphase.
PCT/EP2010/004357 2010-07-16 2010-07-16 Procédé de recyclage d'un biocatalyseur dans un système de solvants à plusieurs constituants à température commandée WO2012007023A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023180310A1 (fr) * 2022-03-21 2023-09-28 Aarhus Universitet Procédé utilisant un système de solvant eutectique profond thermomorphique dans des applications biocatalytiques pour récupérer le biocatalyseur et les produits

Citations (2)

* Cited by examiner, † Cited by third party
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