WO2012107615A1 - Method for producing 1,2-diacetin - Google Patents

Method for producing 1,2-diacetin Download PDF

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Publication number
WO2012107615A1
WO2012107615A1 PCT/ES2012/070059 ES2012070059W WO2012107615A1 WO 2012107615 A1 WO2012107615 A1 WO 2012107615A1 ES 2012070059 W ES2012070059 W ES 2012070059W WO 2012107615 A1 WO2012107615 A1 WO 2012107615A1
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triacetin
diacetin
lipase
hydrolysis
immobilized
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PCT/ES2012/070059
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Spanish (es)
French (fr)
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Roberto FERNÁNDEZ LAFUENTE
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Consejo Superior De Investigaciones Científicas (Csic)
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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
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • Lipases are the enzymes most used in biocatalysis, because they recognize many different substrates but, in many cases, with a high regio- or enantio-selectivity or specificity. In this way, lipases have been used in very different areas of chemistry, for example energy (biodiesel production), food (structured lipid production)) or fine chemistry (for example resolution of racemic mixtures). In addition, lipases are quite robust biocatalysts, which allows their use in unconventional media, such as organic solvents, ionic liquids or supercritical fluids, further increasing the range of possible applications. However, it should not be forgotten that one of the advantages of enzymes compared to other catalysts is that they can be used in totally aqueous media.
  • the natural substrates of lipases are oils and fats.
  • one of the main industrial applications of lipases is the hydrolysis of these natural substrates, usually to obtain free fatty acids or modified oils.
  • Triacetin is apparently a very simple molecule, which can be easily produced by the complete acetylation of glycerol. This molecule is commonly used in lipase activity determinations, but the detailed study of this reaction has not been published, although its regioselective hydrolysis could produce compounds multifunctional and even chiral. For example, regioselective hydrolysis of a single acetyl group of triacetin allows diacetin to be produced, which has a single free hydroxyl group at position 2 or 3. This compound may be useful for some uses, such as the synthesis of O- ( 1,2-di-0-acetyl-glycerol-3-phosphoryl) ethanolamine [1].
  • Reference Ib uses 1,3 regiospecific lipases to achieve 2-monoacetin, which is subsequently chemically acylated to obtain stereospecific diglycerides. But if this hydrolysis occurs only in position 1 or position 3, the carbon 2 of the diacetin will be a chiral center, further increasing the interest of the process.
  • triglycerides usually have a low water solubility as they are formed by long acyl chains, the short triacetin chain significantly increases its water solubility (70 g / L at 25 ° C), allowing hydrolysis in aqueous systems at moderately high concentrations.
  • its low hydrophobicity causes this to be a bad substrate for lipases when it is completely soluble [2-3] and is even quite inefficient in inducing interfacial activation of lipases [4].
  • This patent application describes a regioselective hydrolysis process, preferably in aqueous media, of soluble triacetin to produce 1,2-diacetin in high yields catalyzed by different enzymes, preferably lipases.
  • the reaction is designed so that the migration of acyl groups is minimized and thanks to the different hydrophobicity of diacetin and triacetin, the use of specific enzymes 1, 3, 1,2-diacetin is coupled.
  • this application refers to a process for obtaining 1,2-diacetin characterized in that it comprises performing a hydrolysis of triacetin catalyzed by an enzyme, preferably a lipase, at a pH between 2 and 6, both included.
  • the hydrolysis is carried out at variable temperatures and preferably at a temperature between 4 and 30 ° C.
  • the pH is preferably between 4 and 5.5.
  • the enzyme such as a lipase, for use in this procedure may be immobilized on a support.
  • the support can be by way of example an acrylic support, porous glass, agarose, silica, cellulose, polystyrene, polystyrene divinylbenzene or polyurethane.
  • lipases can be immobilized by physical adsorption, for example, by physical adsorption on hydrophobic supports.
  • the lipase is immobilized as a crosslinked covalent enzyme aggregate or crosslinked enzyme crystal.
  • Useful lipases can be lipases of microorganisms of the genera Candida, Thermomyces, Bacillus, Aspergillus,
  • water-soluble solvents between 10-90% v / v can also be added.
  • Those solvents may be, for example, acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol or propanol.
  • Hydrolysis can also be carried out in a water immiscible solvent, saturated in water.
  • diethyl ether solvent is used.
  • the process comprises the addition of water soluble solvents between 10-90%.
  • v / v which may be selected from, for example, acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol and mixtures thereof.
  • Figure 1 shows the reaction as well as the possible unwanted reactions
  • Figure 2 shows the reaction course of triacetin hydrolysis at pH 5.5 and 22 ° C catalyzed by CALB immobilized in Glutaraldehyde Sepabeads.
  • Figure 3 shows the reaction course of triacetin hydrolysis at pH 5.5 and 22 ° C catalyzed by CALB immobilized in Sepabeads C18. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
  • Figure 4 shows the reaction course of triacetin hydrolysis at pH 5.5 and 22 ° C catalyzed by RML immobilized in Sepabeads C18. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).
  • Figure 5 shows the reaction course of the hydrolysis of triacetin at pH 5.5 and 22 ° C in 20% RML-catalyzed aceonitrile immobilized in Sepabeads C18. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
  • Figure 6 shows the reaction course of the hydrolysis of triacetin at pH 5.5 and 22 ° C in 20% aceonitrile catalyzed by CALB immobilized in Glutaraldehyde Sepabeads. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).
  • acyl migration occurs, obtaining at the moment that all triacetin is consumed a mixture of 1,2- and 1,3-diacetin, in addition to a certain amount of 1 - and 2-monoacetin and even glycerol.
  • PH control is critical. It is recommended to use very concentrated buffers (eg, 1 molar phosphate or acetate) and only raise the pH to 5.5 when the pH drops below pH 3 using a saturated buffer at pH 7 and very good agitation to avoid pH gradients where the migration of the acyl groups will occur
  • very concentrated buffers eg, 1 molar phosphate or acetate
  • reaction slowed down but increased the yields of 1,2-diacetin if miscible co-solvents (5-75% v / v) were used with water (for example acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol, etc. ), possibly by inhibiting the hydrolysis of 1,2 diacetin more strongly than that of triacetin.
  • miscible co-solvents for example acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol, etc.
  • Example 1 Hydrolysis of triacetin at pH 5.5 by Lipase B from Candida Antarctica immobilized on Sepabeads C 18.
  • Glutaraldehyde-Sepabeads was prepared by suspending 10 g of wet EC-HA-Sepabeads support in 20 mL of 5% glutaraldehyde (v / v) in 200 mM phosphate at pH 7.0. The suspension was kept under gentle agitation at 25 ° C for 15 h. After that time, the activated support was filtered and washed thoroughly with distilled water. This treatment was performed to activate all primary support aminos with two glutaraldehyde molecules. The activated support was used immediately after preparation.
  • Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP UV detector 8450) using a Kromasil C18 column (15 cm x 0.46 cm), the retention volumes were 32.0 ml for triacetin, 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin.
  • the concentrations of Triacetmas and both diacetins were calculated using curved reference samples using commercial samples.
  • Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
  • FIG. 2 shows the reaction course of CALB catalyzed triacetin hydrolysis immobilized in Sepabeads glutaraldehyde at pH 5.5 and 22 ° C. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin.)
  • Example 2 Hydrolysis of triacetin at pH 5.5 by Lipase B from Candida Antarctica immobilized on Sepabeads-C 18.
  • a 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC.
  • Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP pump 100 and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column (15 cm x 0.46 was), retention volumes were 32.0 mi for triacetin, 5.8 mi for 1,2-diacetin, 4.8 mi for 1, 3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples.
  • Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
  • FIG. 3 shows the reaction course of CALB catalyzed triacetin hydrolysis immobilized in Sepabeads C18 at pH 5.5 and 22 ° C. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
  • Example 3 Hydrolysis of triacetin at pH 5.5 by Rhizomucor miehei lipase (RML) immobilized on Sepabeads-C 18.
  • RML Rhizomucor miehei lipase
  • a 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC.
  • Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as the mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column (15 cm x 0.46 era), retention volumes were 32.0 mi for triacetin , 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples.
  • Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
  • Figure 4 shows the reaction course of the RML catalyzed hydrolysis of immobilized triacetin in Sepabeads C18 at pH 5.5 and 22 ° C. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).
  • Example 4 Hydrolysis of triacetin at pH 5.5 in the presence of 20% acetonitrile by Rhizomucor miehei lipase (RML) immobilized on Sepabeads-C 18.
  • RML Rhizomucor miehei lipase
  • a 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC.
  • Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column ( 15 cm x 0.46 era), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1.2 diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples.
  • Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
  • Figure 5 shows the reaction course of the hydrolysis of triacetin catalyzed by RML immobilized in Sepabeads C18 at pH 5.5 and 22 ° C in 20% aceonitrile. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
  • Example 5 Hydrolysis of triacetin at pH 5.5 in 20% acetonitrile catalyzed by lipase B from Candida Antarctic immobilized on Sepabeads-glutaraldehyde.
  • Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP UV detector 8450) using a Kromasil C18 column (15 cm x 0.46 cm), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples.
  • Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
  • Figure 6 shows the reaction course of the hydrolysis of triacetin catalyzed by CALB immobilized in Sepabeads glutaraldehyde at pH 5.5 and 22 ° C in 20% aceonitrile. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).

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Abstract

The invention relates to a method for producing 1,2-diacetin, characterized in that it comprises hydrolysis of triacetin catalysed by an enzyme, preferably a lipase, at a pH of between 2 and 6, both inclusive, and at variable temperatures, preferably at a temperature of between 4 and 30°C.

Description

UN PROCEDIMIENTO DE OBTENCIÓN DE 1 ,2-DIACETINA  A PROCEDURE FOR OBTAINING 1,2-DIACETIN
La lipasas son las enzimas más usadas en biocatálisis, porque reconocen muchos sustratos diferentes pero, en muchas ocasiones, con una elevada regio- o enantio-selectividad o especificidad. De esta forma, las lipasas han sido utilizadas en áreas de la química muy diferentes, por ejemplo energía (producción de biodiesel) , alimentación (producción de lípidos estructurados)) o química fina (por ejemplo resolución de mezclas racémicas) . Además, las lipasas son biocatalizadores bastante robustos, lo que permite su empleo en medios no convencionales, como por ejemplo disolventes orgánicos, líquidos iónicos o fluidos supercríticos , aumentando aun más el rango de posibles aplicaciones. Sin embargo, no hay que perder de vista que una de las ventajas de las enzimas comparadas con otros catalizadores es que pueden ser utilizadas en medios totalmente acuosos. Lipases are the enzymes most used in biocatalysis, because they recognize many different substrates but, in many cases, with a high regio- or enantio-selectivity or specificity. In this way, lipases have been used in very different areas of chemistry, for example energy (biodiesel production), food (structured lipid production)) or fine chemistry (for example resolution of racemic mixtures). In addition, lipases are quite robust biocatalysts, which allows their use in unconventional media, such as organic solvents, ionic liquids or supercritical fluids, further increasing the range of possible applications. However, it should not be forgotten that one of the advantages of enzymes compared to other catalysts is that they can be used in totally aqueous media.
Los sustratos naturales de las lipasas son los aceites y grasas. De hecho, una de las principales aplicaciones industriales de las lipasas es la hidrólisis de estos sustratos naturales, normalmente para obtener ácidos grasos libres o aceites modificados. Debido a la naturaleza de sus sustratos, la mayoría de las lipasas presentan un peculiar mecanismo de reacción, con el sitio activo aislado del medio de reacción por una cadena peptídica denominada "lid". En presencia de una superficie hidrofóbica (por ejemplo, una gota de aceite) este lid se mueve para exponer el centro activo y la lipasa se adsorbe a la superficie hidrofóbica de la gota de sustrato.  The natural substrates of lipases are oils and fats. In fact, one of the main industrial applications of lipases is the hydrolysis of these natural substrates, usually to obtain free fatty acids or modified oils. Due to the nature of their substrates, most lipases have a peculiar reaction mechanism, with the active site isolated from the reaction medium by a peptide chain called "lid". In the presence of a hydrophobic surface (for example, a drop of oil) this lid moves to expose the active center and the lipase is adsorbed to the hydrophobic surface of the substrate drop.
La triacetina es aparentemente una molécula muy sencilla, que puede ser fácilmente producida por la completa acetilación del glicerol. Esta molécula se usa habitualmente en determinaciones de actividad de las lipasas, pero el estudio detallado de esta reacción no se ha publicado, a pesar de que su hidrólisis regioselectiva podría producir compuestos multifuncionales e incluso quirales. Por ejemplo, la hidrólisis regioselectiva de un único grupo acetil de la triacetina permite producir diacetina, que tiene un único grupo hidroxilo libre en la posición 2 o 3. Este compuesto puede ser útil para algunos usos, como por ejemplo la síntesis de O- ( 1 , 2-di-0-acetil-glicero-3-fosforil ) etanolamina [1]. Referencia Ib utiliza lipasas 1,3 regioespecí ficas para conseguir 2-monoacetina, que posteriormente se acila químicamente para conseguir diglicéridos estereoespecí fieos . Pero si esta hidrólisis ocurre tan solo en posición 1 o en posición 3, el carbono 2 de la diacetina será un centro quiral, aumentando aún más el interés del proceso. Triacetin is apparently a very simple molecule, which can be easily produced by the complete acetylation of glycerol. This molecule is commonly used in lipase activity determinations, but the detailed study of this reaction has not been published, although its regioselective hydrolysis could produce compounds multifunctional and even chiral. For example, regioselective hydrolysis of a single acetyl group of triacetin allows diacetin to be produced, which has a single free hydroxyl group at position 2 or 3. This compound may be useful for some uses, such as the synthesis of O- ( 1,2-di-0-acetyl-glycerol-3-phosphoryl) ethanolamine [1]. Reference Ib uses 1,3 regiospecific lipases to achieve 2-monoacetin, which is subsequently chemically acylated to obtain stereospecific diglycerides. But if this hydrolysis occurs only in position 1 or position 3, the carbon 2 of the diacetin will be a chiral center, further increasing the interest of the process.
Sin embargo, la migración de los grupos acilos y la racemización puede producir una mezcla de regio y enantioisómeros , disminuyendo el interés del proceso, de forma que hay que seleccionar condiciones experimentales en las que esto no ocurra.  However, the migration of acyl groups and racemization can produce a mixture of regal and enantioisomers, decreasing the interest of the process, so that experimental conditions must be selected in which this does not occur.
Aunque habitualmente los triglicéridos presentan una baja solubilidad en agua al estar formados por largas cadenas de acilo, la corta cadena de la triacetina aumenta significativamente su solubilidad en agua (70 g/L at 25°C) , permitiendo realizar la hidrólisis en sistemas acuosos a concentraciones moderadamente elevadas. Sin embargo, se ha descrito que su baja hidrofobicidad causa que este sea un sustrato malo para lipasas cuando está completamente soluble [2-3] e incluso es bastante poco eficiente en inducir la activación interfacial de lipasas [4] . Incluso en ausencia de migración, en un principio, como se muestra en la referencia Ib, se esperaría que el uso de lipasas en hidrólisis de triacetina condujera a la formación de 2-monoacetina, pasando por una fase de mezcla de 1 , 2-diacetina / 2-monoacetina. Sin embargo, si la triacetina ya no es muy hidrofóbica y no es muy buen sustrato de la enzima, es muy posible que la 1,2 (2,3) diacetina sea aún peor sustrato y en determinadas condiciones pueda conseguirse su acumulación casi total.  Although triglycerides usually have a low water solubility as they are formed by long acyl chains, the short triacetin chain significantly increases its water solubility (70 g / L at 25 ° C), allowing hydrolysis in aqueous systems at moderately high concentrations. However, it has been described that its low hydrophobicity causes this to be a bad substrate for lipases when it is completely soluble [2-3] and is even quite inefficient in inducing interfacial activation of lipases [4]. Even in the absence of migration, initially, as shown in reference Ib, it would be expected that the use of lipases in triacetin hydrolysis would lead to the formation of 2-monoacetin, passing through a 1,2-diacetin mixing phase / 2-monoacetin. However, if triacetin is no longer very hydrophobic and is not a very good enzyme substrate, it is very possible that 1,2 (2,3) diacetin is even worse substrate and under certain conditions its almost total accumulation can be achieved.
Referencias [1] a) Puricelli, L. O- ( 1 , 2di-0-acetyl-glycero-3- phosphoryl ) ethanolamine , a process for its preparation and its therapeutic use. European patent 0 335 331. 1989. References [1] a) Puricelli, L. O- (1, 2di-0-acetyl-glycero-3- phosphoryl) ethanolamine, a process for its preparation and its therapeutic use. European patent 0 335 331. 1989.
b) . Mazur et al . : "REGIO AND STEREOSELECTIVE SYNTHESIS OF TRIGLYCERIDES" W09116442, 1991 b). Mazur et al. : "REGIO AND STEREOSELECTIVE SYNTHESIS OF TRIGLYCERIDES" W09116442, 1991
[2] Sarda, L. Desnuelle P. Action de la lipase pancreatique sur les esters en emulsión Biochim. Biophys. Acta, 1958; 30: 513- 521  [2] Sarda, L. Desnuelle P. Action of the lipase pancreatique sur les esters in Biochim emulsion. Biophys Acta, 1958; 30: 513-521
[3] Ferrato, F. , Carriere, F. , Sarda, L., Verger R. , in:, Methods in Enzymology, B. Rubin, E.A. Dennis (Eds . ) Academic Press, New York. 1997; 286: 327-34  [3] Ferrato, F., Carriere, F., Sarda, L., Verger R., in :, Methods in Enzymology, B. Rubin, E.A. Dennis (Eds.) Academic Press, New York. 1997; 286: 327-34
[4] Pernas, M.A., Pastrana, L . , Fuciños, P., Rúa, M.L. [4] Pernas, M.A., Pastrana, L. , Fuciños, P., Rúa, M.L.
Regulation of the interfacial activation ithin the Candida rugosa lipase family. J. Phys . Org. Chem. , 2009; 22: 508-514. Regulation of the interfacial activation ithin the Candida rugosa lipase family. J. Phys. Org. Chem., 2009; 22: 508-514.
Breve descripción de la invención Brief Description of the Invention
En esta solicitud de patente se describe un procedimiento de hidrólisis regioselectiva, preferentemente en medios acuosos, de triacetina soluble para producir 1,2- diacetina con altos rendimientos catalizada por diferentes enzimas, preferentmente, lipasas. Se diseña la reacción de forma que se minimiza la migración de grupos acilos y gracias a la diferente hidrofobicidad de diacetina y triacetina, al uso de enzimas 1, 3 especificas, se consigue acoplar 1,2- diacetina.  This patent application describes a regioselective hydrolysis process, preferably in aqueous media, of soluble triacetin to produce 1,2-diacetin in high yields catalyzed by different enzymes, preferably lipases. The reaction is designed so that the migration of acyl groups is minimized and thanks to the different hydrophobicity of diacetin and triacetin, the use of specific enzymes 1, 3, 1,2-diacetin is coupled.
Asi esta solicitud se refiere a un procedimiento de obtención de 1 , 2-diacetina caracterizado porque comprende realizar una hidrólisis de triacetina catalizada por un enzima, preferentemente una lipasa, a un pH comprendido entre 2 y 6, ambos valores incluidos.  Thus, this application refers to a process for obtaining 1,2-diacetin characterized in that it comprises performing a hydrolysis of triacetin catalyzed by an enzyme, preferably a lipase, at a pH between 2 and 6, both included.
La hidrólisis se realiza a temperaturas variables y preferentemente a una temperatura comprendida entre 4 y 30 °C.  The hydrolysis is carried out at variable temperatures and preferably at a temperature between 4 and 30 ° C.
Según este procedimiento el pH es preferentemente entre 4 y 5.5.  According to this procedure the pH is preferably between 4 and 5.5.
La enzima, tal como una lipasa, para su uso en este procedimiento puede estar inmovilizada sobre un soporte. Dicho soporte puede ser a modo de ejemplo un soporte acrílico, vidrio poroso, agarosa, sílice, celulosa, poliestireno, poliestireno divinilbenceno o poliuretano. The enzyme, such as a lipase, for use in this procedure may be immobilized on a support. Saying The support can be by way of example an acrylic support, porous glass, agarose, silica, cellulose, polystyrene, polystyrene divinylbenzene or polyurethane.
Además, las lipasas pueden estar inmovilizadas por adsorción física, por ejemplo, por adsorción física sobre soportes hidrofóbicos .  In addition, lipases can be immobilized by physical adsorption, for example, by physical adsorption on hydrophobic supports.
Según otras realizaciones, la lipasa se inmoviliza como agregado enzimático covalente entrecruzado o cristal enzimático entrecruzado.  According to other embodiments, the lipase is immobilized as a crosslinked covalent enzyme aggregate or crosslinked enzyme crystal.
Lipasas útiles pueden ser lipasas de microorganismos de los géneros Candida , Thermomyces , Bacillus, Aspergillus, Useful lipases can be lipases of microorganisms of the genera Candida, Thermomyces, Bacillus, Aspergillus,
Mucor, Rhizomucor . Mucor, Rhizomucor.
Según realizaciones particulara además se pueden añadir disolventes solubles en agua entre el 10-90% v/v. Esos disolventes pueden ser por ejemplo, acetonitrilo, dioxano, tetrahidrofurano , dimetil formamida, metanol, etanol o propanol .  According to particulate embodiments, water-soluble solvents between 10-90% v / v can also be added. Those solvents may be, for example, acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol or propanol.
También se puede realizar la hidrólisis en un disolvente inmiscible con el agua, saturado en agua.  Hydrolysis can also be carried out in a water immiscible solvent, saturated in water.
Según otras realizaciones particulares se usa como disolvente dietil éter.  According to other particular embodiments, diethyl ether solvent is used.
Según otras realizaciones particulares adicionales el procedimiento comprende la adición de disolventes solubles en agua entre el 10-90%. v/v, los cuales pueden estar seleccionados entre, por ejemplo, acetonitrilo, dioxano, tetrahidrofurano , dimetil formamida, metanol, etanol, propanol y mezclas de ellos.  According to other additional particular embodiments the process comprises the addition of water soluble solvents between 10-90%. v / v, which may be selected from, for example, acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol and mixtures thereof.
Breve descripción de las figuras Brief description of the figures
La Figura 1 muestra la reacción así como las posibles reacciones no deseadas Figure 1 shows the reaction as well as the possible unwanted reactions
La figura 2 muestra el curso de reacción de la hidrólisis de triacetina a pH 5,5 y 22°C catalizada por CALB inmovilizada en Sepabeads glutaraldehido . (Círculos: triacetina, Triángulos: diacetina; cuadrados: monoacetina . ) La figura 3 muestra el curso de reacción de la hidrólisis de triacetina a pH 5,5 y 22°C catalizada por CALB inmovilizada en Sepabeads C18. Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina. Figure 2 shows the reaction course of triacetin hydrolysis at pH 5.5 and 22 ° C catalyzed by CALB immobilized in Glutaraldehyde Sepabeads. (Circles: triacetin, Triangles: diacetin; squares: monoacetin.) Figure 3 shows the reaction course of triacetin hydrolysis at pH 5.5 and 22 ° C catalyzed by CALB immobilized in Sepabeads C18. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
La figura 4 muestra el curso de reacción de la hidrólisis de triacetina a pH 5,5 y 22°C catalizada por RML inmovilizada en Sepabeads C18. (Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina) .  Figure 4 shows the reaction course of triacetin hydrolysis at pH 5.5 and 22 ° C catalyzed by RML immobilized in Sepabeads C18. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).
La figura 5 muestra el curso de reacción de la hidrólisis de triacetina a pH 5,5 y 22°C en 20% aceonitrilo catalizada por RML inmovilizada en Sepabeads C18. Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina.  Figure 5 shows the reaction course of the hydrolysis of triacetin at pH 5.5 and 22 ° C in 20% RML-catalyzed aceonitrile immobilized in Sepabeads C18. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
La figura 6 muestra el curso de reacción de la hidrólisis de triacetina a pH 5,5 y 22°C en 20% aceonitrilo catalizada por CALB inmovilizada en Sepabeads glutaraldehido . (Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina) .  Figure 6 shows the reaction course of the hydrolysis of triacetin at pH 5.5 and 22 ° C in 20% aceonitrile catalyzed by CALB immobilized in Glutaraldehyde Sepabeads. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).
Descripción detallada de la invención Detailed description of the invention
Se pretende conseguir frenar la hidrólisis de triacetina catalizada por diferentes lipasas 1,3 especificas (e.g., las de los géneros Candida, Thermomyces, Bacillus, Aspergillus , Mucor, Rhizomucor) de forma libre o inmovilizada en diferentes soportes (e.g. soportes acrilicos, vidrio poroso, agarosa, sílice, celulosa, poliestireno , poliestireno divinilbenceno , poliuretano) , bien de forma covalente o bien por absorción física, en 1 , 2-diacetina (figura 1), para su posterior empleo en el diseño de diferentes productos regio y enantioespecífieos .  It is intended to stop the hydrolysis of triacetin catalyzed by different specific 1,3 lipases (eg, those of the genera Candida, Thermomyces, Bacillus, Aspergillus, Mucor, Rhizomucor) freely or immobilized on different supports (eg acrylic supports, porous glass , agarose, silica, cellulose, polystyrene, polystyrene divinylbenzene, polyurethane), either covalently or by physical absorption, in 1, 2-diacetin (Figure 1), for later use in the design of different regal and enantiospecific products.
A pH 7 y 4°C o 25°C se produce la migración de acilos obteniéndose en el momento en el que se consume toda la triacetina una mezcla de 1,2- y 1 , 3-diacetina, además de una cierta cantidad de 1- y 2-monoacetina e incluso glicerol.  At pH 7 and 4 ° C or 25 ° C, acyl migration occurs, obtaining at the moment that all triacetin is consumed a mixture of 1,2- and 1,3-diacetin, in addition to a certain amount of 1 - and 2-monoacetin and even glycerol.
Sin embargo en el intervalo de pH 6-3 y en el intervalo de temperaturas de 4 a 37 °C se consigue evitar la formación de 1 , 3-diacetina por migración y además se produce la acumulación de 1 , 2-diacetina, con muy alto rendimiento (por encima del 80% e incluso cercano al 100%), para todas las preparaciones de las diferentes lipasas utilizadas. Los resultados no se afectaban apenas por el uso de diferentes concentraciones de triacetina, desde 100 mM (en el que la triacetina es totalmente soluble) a 5 M de triacetina (en este caso se forma un sistema bifásico con parte de la triacetina soluble y el resto en una fase insoluble triacetina, que se va consumiendo hasta producir entre un 80% y > 98% de 1 , 2-diacetina) . However, in the range of pH 6-3 and in the temperature range of 4 to 37 ° C it is possible to avoid the formation of 1,3-diacetin by migration and in addition the accumulation of 1,2-diacetin occurs, with very high performance (above 80% and even close to 100%), for all preparations of the different lipases used. The results were hardly affected by the use of different concentrations of triacetin, from 100 mM (in which triacetin is completely soluble) to 5 M triacetin (in this case a biphasic system with part of the soluble triacetin is formed and the rest in an insoluble phase triacetin, which is consumed until producing between 80% and> 98% of 1,2-diacetin).
El control del pH es critico. Se recomienda utilizar tampones muy concentrados (e.g., 1 molar de fosfato o de acetato) y solo subir el pH hasta 5.5 cuando el pH desciende por debajo de pH 3 utilizando un tampón saturado a pH 7 y muy buena agitación para evitar gradientes de pH donde se producirá la migración de los grupos acilos  PH control is critical. It is recommended to use very concentrated buffers (eg, 1 molar phosphate or acetate) and only raise the pH to 5.5 when the pH drops below pH 3 using a saturated buffer at pH 7 and very good agitation to avoid pH gradients where the migration of the acyl groups will occur
La reacción bajaba en velocidad pero aumentaba los rendimientos de 1 , 2-diacetina si se utilizan codisolventes miscible (5-75% v/v) con agua (por ejemplo acetonitrilo, dioxano, tetrahidrofurano , dimetil formamida, metanol, etanol, propanol, etc) , posiblemente al inhibir de forma más acusada la hidrólisis de la 1,2 diacetina que al de triacetina.  The reaction slowed down but increased the yields of 1,2-diacetin if miscible co-solvents (5-75% v / v) were used with water (for example acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol, etc. ), possibly by inhibiting the hydrolysis of 1,2 diacetin more strongly than that of triacetin.
Se ensayó también la reacción de hidrólisis en dietil éter saturado en agua, un disolvente descrito como inhibidor de la migración de acilos. En este caso se utilizaron solo enzimas inmovilizadas o agregadas, el disolvente está saturado previamente en tampón acetato a pH 5, añadiéndose progresivamente este tampón a lo largo de la reacción para compensar el agua consumida por la hidrólisis. La velocidad de reacción fue menor con la mayoría de las preparaciones de lipasas, pero los rendimientos conseguidos de 1,2 diacetina fueron similares (80- >98%) .  The hydrolysis reaction in diethyl ether saturated in water, a solvent described as an inhibitor of acyl migration was also tested. In this case only immobilized or aggregated enzymes were used, the solvent is previously saturated in acetate buffer at pH 5, this buffer being added progressively throughout the reaction to compensate for the water consumed by hydrolysis. The reaction rate was lower with most lipase preparations, but the yields of 1.2 diacetin were similar (80-> 98%).
Para todos los casos ensayados, usando las diferentes lipasas descritas anteriormente libres o inmovilizadas en diferentes soportes, en condiciones donde la migración de acilo era disminuida o eliminada se conseguían rendimientos superiores al 80% de 1 , 2-diacetina . Ejemplos de la invención For all the cases tested, using the different lipases described above free or immobilized on different supports, under conditions where the acyl migration was reduced or eliminated yields greater than 80% of 1,2-diacetin were achieved. Examples of the invention
Ejemplo 1. Hidrólisis de triacetina a pH 5.5 por la lipasa B de Candida antárctica inmovilizada sobre Sepabeads C 18. Example 1. Hydrolysis of triacetin at pH 5.5 by Lipase B from Candida Antarctica immobilized on Sepabeads C 18.
Glutaraldehído-Sepabeads fue preparado suspendiendo 10 g de soporte EC-HA-Sepabeads húmedo en 20 mL del5% glutaraldehido (v/v) en fosfato 200 mM a pH 7.0. La suspensión se mantuvo bajo agitación suave a 25°C durante 15 h. Después de ese tiempo, el soporte activado se filtró y se lavó exhaustivamente con agua destilada. Este tratamiento se realizó para activar todos los aminos primarios del soporte con dos moléculas de glutaraldehido. El soporte activado se utilizó inmediatamente después de su preparación.  Glutaraldehyde-Sepabeads was prepared by suspending 10 g of wet EC-HA-Sepabeads support in 20 mL of 5% glutaraldehyde (v / v) in 200 mM phosphate at pH 7.0. The suspension was kept under gentle agitation at 25 ° C for 15 h. After that time, the activated support was filtered and washed thoroughly with distilled water. This treatment was performed to activate all primary support aminos with two glutaraldehyde molecules. The activated support was used immediately after preparation.
10 g de glutaraldehido-Sepabeads se añadieron a 400 mL de una disolución de lipasa (0,5 mg/mL) en fosfato sódico 10 mM a pH 7 en presencia de 0.1 % (v/v) Tritón X -100. Tras 7 horas a 22 °C bajo agitación a 250 rpm, la suspensión se filtro y la lipasa inmovilizada se lavó 5 veces con 10 volúmenes de agua destilada, el biocatalizador húmedo se almacenó a 4°C.  10 g of glutaraldehyde-Sepabeads were added to 400 mL of a solution of lipase (0.5 mg / mL) in 10 mM sodium phosphate at pH 7 in the presence of 0.1% (v / v) Triton X -100. After 7 hours at 22 ° C under stirring at 250 rpm, the suspension was filtered and the immobilized lipase was washed 5 times with 10 volumes of distilled water, the wet biocatalyst was stored at 4 ° C.
Por otro lado se preparó una disolución de 100 mM de triacetina en 500 mM de fosfato sódico y su pH se ajustó a pH 5.5. El sustrato era completamente soluble en estas condiciones .  On the other hand, a solution of 100 mM of triacetin in 500 mM of sodium phosphate was prepared and its pH adjusted to pH 5.5. The substrate was completely soluble under these conditions.
Se añade una muestra de 1,5 g de biocatalizador húmedo a 40 mL de la disolución de triacetina y las suspensiones de reacción se agitaron suavemente en un agitador a 250 rpm y 22°C. Periódicamente se sacaron muestras de esta suspensión, se descartó el biocatalizador por centrifugación y la concentración de los productos de reacción se analizó por HPLC. Diacetina y triacetina se analizaron usando 10% acetonitrilo/ 90% agua (v/v) como fase móvil a un flujo de 1 ml/min y una columna RP-HPLC (con una bomba Spectra Physic SP 100 y un detector UV Spectra Physic SP 8450) usando una columna Kromasil C18 (15 cm x 0,46 cm) , los volúmenes de retención fueron 32.0 mi para triacetina, 5,8 mi para 1,2- diacetina, 4.8 mi para 1 , 3-diacetina . Las concentraciones de triacetmas y ambas diacetinas se calcularon usando muestras curvas de referencia usando muestras comerciales. Monoacetina se analizó usando la misma columna y flujo, pero la fase móvil, fue 1,5% acetonitrilo/ 98,5% agua (v/v) , con un volumen de retención de 3.6 mi mientras el ácido acético eluye en 3.0 mi. A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred on a shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP UV detector 8450) using a Kromasil C18 column (15 cm x 0.46 cm), the retention volumes were 32.0 ml for triacetin, 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin. The concentrations of Triacetmas and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
Tras 2 horas de reacción se habla consumido toda la triacetina, con un 95% de de 1 , 2-diacetina y un 5% de 2- monoacetina. El catalizador pudo reutilizarse en 5 ciclos sin que se observase variación en velocidad o rendimiento. La figura 2 muestra el curso de reacción de la hidrólisis de triacetina catalizada por CALB inmovilizada en Sepabeads glutaraldehido at pH 5,5 y 22°C. (Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina.)  After 2 hours of reaction, all triacetin has been consumed, with 95% of 1,2-diacetin and 5% of 2-monoacetin. The catalyst could be reused in 5 cycles without any variation in speed or performance. Figure 2 shows the reaction course of CALB catalyzed triacetin hydrolysis immobilized in Sepabeads glutaraldehyde at pH 5.5 and 22 ° C. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin.)
Ejemplo 2. Hidrólisis de triacetina a pH 5.5 por la lipasa B de Candida antárctica inmovilizada sobre Sepabeads-C 18. Example 2. Hydrolysis of triacetin at pH 5.5 by Lipase B from Candida Antarctica immobilized on Sepabeads-C 18.
10 gramos de octadecil-Sepabeads se añadieron a 400 mL de CALB en una disolución de lipasa (0.5 mg/mL) en tampón fosfato 10 mM a pH 7 y 22°C. Después de 7 h bajo agitación a 250 rpm, la suspensión se filtro y la lipasa se lavó 5 veces con 10 volúmenes de agua destilada. La preparación húmeda se almacenó a 4°C.  10 grams of octadecyl-Sepabeads were added to 400 mL of CALB in a lipase solution (0.5 mg / mL) in 10 mM phosphate buffer at pH 7 and 22 ° C. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 10 volumes of distilled water. The wet preparation was stored at 4 ° C.
Por otro lado se preparó una disolución de 100 mM de triacetina en 500 mM de fosfato sódico y su pH se ajustó a pH 5.5. El sustrato era completamente soluble en estas condiciones .  On the other hand, a solution of 100 mM of triacetin in 500 mM of sodium phosphate was prepared and its pH adjusted to pH 5.5. The substrate was completely soluble under these conditions.
Se añade una muestra de 1,5 g de biocatalizador húmedo a 40 mL de la disolución de triacetina y las suspensiones de reacción se agitaron suavemente en un Shaker a 250 rpm y 22 °C. Periódicamente se sacaron muestras de esta suspensión, se descartó el biocatalizador por centrifugación y la concentración de los productos de reacción se analizó por HPLC. Diacetina y triacetina se analizaron usando 10% acetonitrilo/ 90% agua (v/v) como fase móvil a un flujo de 1 ml/min y una columna RP-HPLC (con una bomba Spectra Physic SP 100 y un detector UV Spectra Physic SP 8450) usando una columna Kromasil C18 (15 cm x 0,46 era) , volúmenes de retención fueron 32.0 mi para triacetina, 5,8 mi para 1 , 2-diacetina, 4.8 mi para 1 , 3-diacetina . Las concentraciones de triacetinas y ambas diacetinas se calcularon usando muestras curvas de referencia usando muestras comerciales. Monoacetina se analizó usando la misma columna y flujo, pero la fase móvil, fue 1,5% acetonitrilo/ 98,5% agua (v/v), con un volumen de retención de 3.6 mi mientras el ácido acético eluye en 3.0 mi. A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP pump 100 and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column (15 cm x 0.46 was), retention volumes were 32.0 mi for triacetin, 5.8 mi for 1,2-diacetin, 4.8 mi for 1, 3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
Tras 60 minutos de reacción se agota la triacetina y se consigue un rendimiento del 87% de 1,2 diacetina y 13% de 2- monoacetina. La figura 3 muestra el curso de reacción de la hidrólisis de triacetina catalizada por CALB inmovilizada en Sepabeads C18 at pH 5,5 y 22°C. Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina.  After 60 minutes of reaction, triacetin is depleted and a yield of 87% of 1.2 diacetin and 13% of 2-monoacetin is achieved. Figure 3 shows the reaction course of CALB catalyzed triacetin hydrolysis immobilized in Sepabeads C18 at pH 5.5 and 22 ° C. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
Ejemplo 3. Hidrólisis de triacetina a pH 5.5 por la lipasa de Rhizomucor miehei (RML) inmovilizada sobre Sepabeads-C 18. Example 3. Hydrolysis of triacetin at pH 5.5 by Rhizomucor miehei lipase (RML) immobilized on Sepabeads-C 18.
10 gramos de octadecil-Sepabeads se añadieron a 55 mL (RML) de una disolución de lipasa (0.5 mg/mL) en tampón fosfato 10 mM a pH 7 y 22°C. Después de 7 h bajo agitación a 250 rpm, la suspensión se filtró y la lipasa se lavó 5 veces con 10 volúmenes de agua destilada. La preparación húmeda se almacenó a 4°C.  10 grams of octadecyl-Sepabeads were added to 55 mL (RML) of a lipase solution (0.5 mg / mL) in 10 mM phosphate buffer at pH 7 and 22 ° C. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 10 volumes of distilled water. The wet preparation was stored at 4 ° C.
Por otro lado se preparó una disolución de 100 mM de triacetina en 500 mM de fosfato sódico y su pH se ajustó a pH 5.5. El sustrato era completamente soluble en estas condiciones .  On the other hand, a solution of 100 mM of triacetin in 500 mM of sodium phosphate was prepared and its pH adjusted to pH 5.5. The substrate was completely soluble under these conditions.
Se añade una muestra de 1,5 g de biocatalizador húmedo a 40 mL de la disolución de triacetina y las suspensiones de reacción se agitaron suavemente en un Shaker a 250 rpm y 22°C. Periódicamente se sacaron muestras de esta suspensión se descarto el biocatalizador por centrifugación y la concentración de los productos de reacción se analizó por HPLC. Diacetina y triacetina se analizaron usando 10% acetonitrilo/ 90% agua (v/v) como fase móvil a un flujo de 1 ml/min y una columna RP-HPLC (con una bomba Spectra Physic SP 100 y un detector UV Spectra Physic SP 8450) usando una columna Kromasil C18 (15 cm x 0,46 era) , los volúmenes de retención fueron 32.0 mi para triacetina, 5,8 mi para 1,2- diacetina, 4.8 mi para 1 , 3-diacetina . Las concentraciones de triacetinas y ambas diacetinas se calcularon usando muestras curvas de referencia usando muestras comerciales. Monoacetina se analizó usando la misma columna y flujo, pero la fase móvil fue 1,5% acetonitrilo/ 98,5% agua (v/v) , con un volumen de retención de 3.6 mi mientras el ácido acético eluye en 3.0 mi. A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as the mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column (15 cm x 0.46 era), retention volumes were 32.0 mi for triacetin , 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
Tras 1 hora se consigue un 88% de 1,2 diacetina y un 12% de 2-monoacetina . La figura 4 muestra el curso de reacción de la hidrólisis de triacetina catalizada por RML inmovilizada en Sepabeads C18 at pH 5,5 y 22°C. (Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina) .  After 1 hour, 88% of 1.2 diacetin and 12% of 2-monoacetin are achieved. Figure 4 shows the reaction course of the RML catalyzed hydrolysis of immobilized triacetin in Sepabeads C18 at pH 5.5 and 22 ° C. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).
Ejemplo 4. Hidrólisis de triacetina a pH 5.5 en presencia de 20% acetonitrilo por la lipasa de Rhizomucor miehei (RML) inmovilizada sobre Sepabeads-C 18. Example 4. Hydrolysis of triacetin at pH 5.5 in the presence of 20% acetonitrile by Rhizomucor miehei lipase (RML) immobilized on Sepabeads-C 18.
10 gramos de octadecil-Sepabeads se añadieron a 55 mL (RML) de una disolución de lipasa (0.5 mg/mL) en tampón fosfato 10 mM a pH 7 y 22°C. Después de 7 h bajo agitación a 250 rpm, la suspensión se filtró y la lipasa se lavó 5 veces con 10 volúmenes de agua destilada. La preparación húmeda se almacenó a 4°C.  10 grams of octadecyl-Sepabeads were added to 55 mL (RML) of a lipase solution (0.5 mg / mL) in 10 mM phosphate buffer at pH 7 and 22 ° C. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 10 volumes of distilled water. The wet preparation was stored at 4 ° C.
Por otro lado se preparó una disolución de 100 mM de triacetina en 500 mM de fosfato sódico /20% acetonitrilo (v/v) y su pH se ajustó a pH 5.5. El sustrato era completamente soluble en estas condiciones.  On the other hand, a solution of 100 mM triacetin in 500 mM sodium phosphate / 20% acetonitrile (v / v) was prepared and its pH adjusted to pH 5.5. The substrate was completely soluble under these conditions.
Se añade una muestra de 1,5 g de biocatalizador húmedo a 40 mL de la disolución de triacetina y las suspensiones de reacción se agitaron suavemente en un Shaker a 250 rpm y 22°C. Periódicamente se sacaron muestras de esta suspensión, se descartó el biocatalizador por centrifugación y la concentración de los productos de reacción se analizó por HPLC. Diacetina y triacetina se analizaron usando 10% acetonitrilo/ 90% agua (v/v) como fase móvil a un flujo de 1 ml/min y una columna RP-HPLC (con una bomba Spectra Physic SP 100 y un detector UV Spectra Physic SP 8450) usando una columna Kromasil C18 (15 cm x 0,46 era) , volúmenes de retención fueron 32.0 mi para triacetina, 5,8 mi para 1,2 diacetina, 4.8 mi para 1 , 3-diacetina . Las concentraciones de triacetinas y ambas diacetinas se calcularon usando muestras curvas de referencia usando muestras comerciales. Monoacetina se analizó usando la misma columna y flujo, pero la fase móvil, fue 1,5% acetonitrilo/ 98,5% agua (v/v), con un volumen de retención de 3.6 mi mientras el ácido acético eluye en 3.0 mi. A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the concentration of the reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP 8450 UV detector) using a Kromasil C18 column ( 15 cm x 0.46 era), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1.2 diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
Tras 4 h de reacción, el rendimiento supera el 95%.  After 4 h of reaction, the yield exceeds 95%.
La figura 5 muestra el curso de reacción de la hidrólisis de triacetina catalizada por RML inmovilizada en Sepabeads C18 at pH 5,5 y 22°C en 20% aceonitrilo. Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina. Figure 5 shows the reaction course of the hydrolysis of triacetin catalyzed by RML immobilized in Sepabeads C18 at pH 5.5 and 22 ° C in 20% aceonitrile. Circuits: triacetin, Triangles: diacetin; Squares: monoacetin.
Ejemplo 5. Hidrólisis de triacetina a pH 5.5 en 20% acetonitrilo catalizada por la lipasa B de Candida antárctica inmovilizada sobre Sepabeads-glutaraldehido . Example 5. Hydrolysis of triacetin at pH 5.5 in 20% acetonitrile catalyzed by lipase B from Candida Antarctic immobilized on Sepabeads-glutaraldehyde.
10 gramos de octadecil-Sepabeads se añadieron a 400 mL de CALB en una disolución de lipasa (0.5 mg/mL) en tampón fosfato 10 mM a pH 7 y 22°C. Después de 7 h bajo agitación a 250 rpm, la suspensión se filtró y la lipasa se lavó 5 veces con 10 volúmenes de agua destilada. La preparación húmeda se almacenó a 4°C.  10 grams of octadecyl-Sepabeads were added to 400 mL of CALB in a lipase solution (0.5 mg / mL) in 10 mM phosphate buffer at pH 7 and 22 ° C. After 7 h under stirring at 250 rpm, the suspension was filtered and the lipase was washed 5 times with 10 volumes of distilled water. The wet preparation was stored at 4 ° C.
Por otro lado se preparó una disolución de 100 mM de triacetina en 500 mM de fosfato sódico y su pH se ajustó a pH 5.5. El sustrato era completamente soluble en estas condiciones .  On the other hand, a solution of 100 mM of triacetin in 500 mM of sodium phosphate was prepared and its pH adjusted to pH 5.5. The substrate was completely soluble under these conditions.
Se añade una muestra de 1,5 g de biocatalizador húmedo a 40 mL de la disolución de triacetina y las suspensiones de reacción se agitaron suavemente en un Shaker a 250 rpm y 22°C. Periódicamente se sacaron muestras de esta suspensión se descarto el biocatalizador por centrifugación y la concentración de los productos de reacción se analizó por HPLC. Diacetina y triacetina se analizaron usando 10% acetonitrilo/ 90% agua (v/v) como fase móvil a un flujo de 1 ml/min y una columna RP-HPLC (con una bomba Spectra Physic SP 100 y un detector UV Spectra Physic SP 8450) usando una columna Kromasil C18 (15 cm x 0,46 cm) , volúmenes de retención fueron 32.0 mi para triacetina, 5,8 mi para 1 , 2-diacetina, 4.8 mi para 1 , 3-diacetina . Las concentraciones de triacetinas y ambas diacetinas se calcularon usando muestras curvas de referencia usando muestras comerciales. Monoacetina se analizo usando la misma columna y flujo, pero la fase móvil, fue 1,5% acetonitrilo/ 98,5% agua (v/v), con un volumen de retención de 3.6 mi mientras el ácido acético eluye en 3.0 mi. A 1.5 g sample of wet biocatalyst is added to 40 mL of the triacetin solution and the reaction suspensions are gently stirred in a Shaker at 250 rpm and 22 ° C. Samples were periodically taken from this suspension, the biocatalyst was discarded by centrifugation and the Concentration of reaction products was analyzed by HPLC. Diacetin and triacetin were analyzed using 10% acetonitrile / 90% water (v / v) as a mobile phase at a flow of 1 ml / min and an RP-HPLC column (with a Spectra Physic SP 100 pump and a Spectra Physic SP UV detector 8450) using a Kromasil C18 column (15 cm x 0.46 cm), retention volumes were 32.0 ml for triacetin, 5.8 ml for 1,2-diacetin, 4.8 ml for 1,3-diacetin. The concentrations of triacetins and both diacetins were calculated using curved reference samples using commercial samples. Monoacetin was analyzed using the same column and flow, but the mobile phase was 1.5% acetonitrile / 98.5% water (v / v), with a retention volume of 3.6 ml while acetic acid eluted at 3.0 ml.
Tras 2 un 97% de 1,2 diacetina podia ser observado con un 1% de triacetina aun presente en el medio de reacción, a las 3 h un 98% de diacetina podia ser observado y a las 4 aun se mantiene un 87%. La figura 6 muestra el curso de reacción de la hidrólisis de triacetina catalizada por CALB inmovilizada en Sepabeads glutaraldehido at pH 5,5 y 22°C en 20% aceonitrilo. (Circuios: triacetina, Triángulos: diacetina; cuadrados: monoacetina) .  After 2 97% of 1.2 diacetin could be observed with 1% triacetin still present in the reaction medium, at 3 h 98% of diacetin could be observed and at 4 o'clock it is still maintained 87%. Figure 6 shows the reaction course of the hydrolysis of triacetin catalyzed by CALB immobilized in Sepabeads glutaraldehyde at pH 5.5 and 22 ° C in 20% aceonitrile. (Circuits: triacetin, Triangles: diacetin; squares: monoacetin).

Claims

Reivindicaciones Claims
1. Un procedimiento de obtención de 1 , 2-diacetina caracterizado porque comprende realizar una hidrólisis de triacetina catalizada por un enzima a un pH comprendido entre 2 y 6, ambos valores incluidos. 1. A process for obtaining 1,2-diacetin characterized in that it comprises performing a hydrolysis of triacetin catalyzed by an enzyme at a pH between 2 and 6, both included.
2. Un procedimiento según la reivindicación 1, caracterizado porque el enzima es una lipasa.  2. A method according to claim 1, characterized in that the enzyme is a lipase.
3. Un procedimiento según la reivindicación 1, caracterizado porque se realiza a una temperatura comprendida entre 4 y 30°C.  3. A method according to claim 1, characterized in that it is carried out at a temperature between 4 and 30 ° C.
4. Un procedimiento según la reivindicación 1, caracterizado porque comprende hidrolizar triacetina a un pH de entre 4 y 5.5.  4. A process according to claim 1, characterized in that it comprises hydrolyzing triacetin at a pH between 4 and 5.5.
5. Un procedimiento según la reivindicación 1 Ó 2, caracterizado porque la lipasa está inmovilizada sobre un soporte .  5. A method according to claim 1 or 2, characterized in that the lipase is immobilized on a support.
6. Un procedimiento según la reivindicación 5, caracterizado porque el soporte está seleccionado entre soportes acrílicos, vidrio poroso, agarosa, sílice, celulosa, poliestireno , poliestireno divinilbenceno y poliuretano.  A process according to claim 5, characterized in that the support is selected from acrylic supports, porous glass, agarose, silica, cellulose, polystyrene, polystyrene divinylbenzene and polyurethane.
7. Un procedimiento según la reivindicación 5, caracterizado porque la hidrólisis se realiza utilizando lipasas inmovilizadas por adsorción física.  7. A process according to claim 5, characterized in that the hydrolysis is carried out using lipases immobilized by physical adsorption.
8. Un procedimiento según la reivindicación 7, caracterizado porque la hidrólisis se realiza utilizando lipasas inmovilizadas por adsorción física sobre soportes hidrofóbicos .  A method according to claim 7, characterized in that the hydrolysis is carried out using lipases immobilized by physical adsorption on hydrophobic supports.
9. Un procedimiento según la reivindicación 5, caracterizado porque la enzima es una lipasa y se inmoviliza como agregado enzimático covalente entrecruzado o cristal enzimático entrecruzado.  9. A method according to claim 5, characterized in that the enzyme is a lipase and is immobilized as a cross-linked covalent enzyme aggregate or cross-linked enzyme crystal.
10. Un procedimiento, según la reivindicación 2, caracterizado porque la lipasa está seleccionada entre lipasas de microorganismos de los géneros Candida , Thermomyces, Bacillus , Aspergillus, Mucor y Rhizomucor . 10. A method according to claim 2, characterized in that the lipase is selected from lipases of microorganisms of the genera Candida, Thermomyces, Bacillus, Aspergillus, Mucor and Rhizomucor.
11. Un procedimiento según la reivindicación 1, caracterizado porque comprende además adición de disolventes solubles en agua en una cantidad comprendida entre el 10-90% v/v . A method according to claim 1, characterized in that it further comprises adding water soluble solvents in an amount between 10-90% v / v.
12. Un procedimiento según la reivindicación 11, caracterizado porque dichos disolventes están seleccionados entre acetonitrilo, dioxano, tetrahidrofurano , dimetil formamida, metanol, etanol y propanol.  12. A process according to claim 11, characterized in that said solvents are selected from acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol and propanol.
13. Un procedimiento según la reivindicación 1, caracterizado porque se realiza en un disolvente inmiscible con el agua, saturado en agua.  13. A process according to claim 1, characterized in that it is carried out in a solvent immiscible with water, saturated in water.
14. Un procedimiento según la reivindicación 13, caracterizado porque se usa como disolvente dietil éter.  14. A process according to claim 13, characterized in that diethyl ether is used as solvent.
15. Un procedimiento según la reivindicación 13, caracterizado porque comprende además adición de disolventes solubles en agua en una cantidad comprendida entre el 10-90%. v/v .  15. A process according to claim 13, characterized in that it further comprises the addition of water-soluble solvents in an amount between 10-90%. v / v.
16. Un procedimiento según la reivindicación 13, caracterizado porque dichos disolventes están seleccionados entre acetonitrilo, dioxano, tetrahidrofurano , dimetil formamida, metanol, etanol, propanol y mezclas de ellos.  16. A process according to claim 13, characterized in that said solvents are selected from acetonitrile, dioxane, tetrahydrofuran, dimethyl formamide, methanol, ethanol, propanol and mixtures thereof.
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