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    • 专利摘要:
    Enzymatic esterification and/or transesterification procedure using supported enzymatic biocatalysts. Process for the esterification and/or transesterification of oleins characterized in that it comprises performing the esterification and/or transesterification with a biocatalyst supported on a magnetic nano-support covalently anchored to it, and a method of preparing the supported biocatalyst that comprises the following steps: 1. Preparation of a magnetic nanocarrier, 2. surface activation of the nanocarrier, 3. construction of a bridging ligand and 4. covalent anchoring of the enzyme. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2804057A1
    申请号:ES201930697
    申请日:2019-07-29
    公开日:2021-02-02
    发明作者:Esteban Alejandro Victor Martinez；Julian Luis Francisco Martin；Zamorano Laura Sanchez
    申请人:Asociacion de la Ind Navarra Ain；
    IPC主号:
    专利说明:

    [0001] ENZYMATICS THROUGH SUPPORTED ENZYMATIC BIOCATALIZERS
    [0002] FIELD OF THE INVENTION
    [0004] The present invention relates to an enzymatic esterification and / or transesterification process supported on a magnetic nano-support in order to allow the reuse of the biocatalyst. This supported biocatalyst has been used in the recovery of oleins. The products resulting from this process have multiple uses in the chemical, cosmetic, pharmaceutical and food industries.
    [0006] BACKGROUND
    [0008] The obtaining of monoglycerides (MAG) and triglycerides (TAG) from industrial oleaginous waste has been carried out with the use of a chemical technology that requires high processing temperatures (190-250 ° C), basic pH and subsequent purification processes. . Enzyme technology has many advantages over chemical technology, for example, the working temperature is lower (50-80 ° C), the working pH is close to neutral (pH 5.0-6.0) which makes it a Much less aggressive technology with the raw material, the final product and the environment due to the generation of lower amounts of waste in addition to the use of fewer energy resources in its manufacture. However, enzymatic technology is expensive due to the high price of the free biocatalysts used and because these biocatalysts cannot be reused. For this reason, a development that allows the reuse of a biocatalyst for a process of these characteristics (esterification / transesterification of oleaginous by-products of vegetable origin, although they could be others including non-waste raw materials) represents a cheaper process. of current glycerolysis and an improvement of the products obtained compared to processes with chemical technology (G. Fernández-Lorente et al. Biomacromolecules 2006, 7, 2610, L. Betancor et al. Enzyme and Microbial Technology 2006, 39, 877, K. Can et al. Colloids and Surfaces B: Biointerfaces 2009, 71, 154, Y. Liu et al. Langmuir 2013, 29, 15275, MK Naik et al. Catalysis Today 2014, 237, 145, A. García Soleasa et al. Food Chemistry 2016, 190, 960).
    [0010] Patent ES2159231 B1 discloses a method for the selective esterification of alpha primary and secondary alcohols in the presence of a biocatalyst comprising one or more lipases, which can be supported on a resin, but does not describe whether the enzyme is bound to a nanocarrier, nor that the enzyme is recoverable.
    [0011] In the present invention, an enzyme-catalyzed esterification and / or transesterification process has been developed where the supported biocatalyst is covalently anchored to a magnetic nano-support. The objective of this anchoring is twofold: On the one hand, supported biocatalysts have the advantage of greater ease of recovery, recycling and reuse than free catalysts. On the other hand, the covalent bonding of a biocatalyst to a magnetic nano-support improves its thermal stability and catalytic activity. The improvement in catalytic activity, a key parameter of this invention, is generated by inducing a rigidity in the morphology of the biocatalyst that forces it to remain in its active conformation (JM Guisán et al. Biomacromolecules 2006, 7, 2610. P. Adlercreutz. Chem. Soc. Rev.
    [0012] 2013, 42, 6406).
    [0014] DESCRIPTION
    [0016] As used herein the term "olein" is a term that refers to acidic oils.
    [0017] As used herein the terms "biocatalyst" and "enzyme" are synonymous and are used interchangeably.
    [0019] In the present specification the term "neutralization" is understood as the approximation of the pH of the oleins to the optimum pH of action of the biocatalyst.
    [0021] As used herein, "magnetic nanocarrier" means a nanostructure with superparamagnetic properties.
    [0023] Biocatalysts are the fundamental tool of this project. Due to its high cost, the need arises to develop a heterogeneous (supported) catalyst to make the process more competitive and to be able to give it more uses.
    [0025] The present invention discloses a process for the esterification and / or transesterification of oleins characterized in that it comprises carrying out the esterification and / or transesterification with a biocatalyst supported on a magnetic nanocarrier covalently anchored thereto.
    [0027] In a particular embodiment, said esterification and / or transesterification process is glycerolysis.
    [0029] In a particular embodiment, said process further comprises optimizing the pH of the oleins. Said pH is between 5.5 and 6.5.
    [0031] Any base can be used to optimize said pH, in particular NaOH.
    [0032] The method of the present invention according to particular embodiments comprises:
    [0034] - arrange the oleins with a pH between 5.5 and 6.5 and with a water content of less than 2% and add, in this order:
    [0036] - alkyl alcohols and / or polyalcohols, for example glycerol, and
    [0038] - the supported biocatalyst
    [0040] and carrying out the esterification / transesterification reaction at a temperature between 50 and 70 ° C, preferably 65 ° C.
    [0042] The amount of supported biocatalyst is comprised between 0.5% and 2% of the sum of the mass of alkyl alcohols and / or polyalcohols and oleins. The amount of supported biocatalyst is in the range of 0.2-5% of the total of the reaction mixture.
    [0044] The biocatalyst is a lipase and more preferably CALB lipase.
    [0046] According to preferred embodiments, the ratio between oleins and alkyl alcohols and / or polyalcohols is 1.5: 1 to 4: 1, more preferably 1.5: 1 to 2.5: 1.
    [0048] The esterification / transesterification reaction proceeds under vacuum at a pressure within the range of 0-50 millibars (mbar), preferably 0 mbar.
    [0050] The higher the pressure, the lower the reaction yield. The function of the vacuum is to remove volatile by-products that are generated in the reaction, and that slow it down or force equilibrium.
    [0052] The esterification / transesterification reaction proceeds at a stirring of between 100 and 1000 revolutions per minute (rpm), preferably 700 rpm. The objective is that a turbulent regime is generated in the reactor so that the interface area, in which the reaction is carried out, is maximum
    [0054] The reaction is carried out in a time comprised between 15-30hr, preferably 24hr.
    [0056] According to preferred embodiments of the process, the magnetic nanocarriers are superparamagnetic nanostructures of Fe.
    [0057] Obtaining supported biocatalysts
    [0059] The method for the preparation of the supported biocatalyst is adapted from protocols described in the bibliography with some modifications (M. Bilal et al. International Journal of Biological Macromolecules 2018, 120, 2530, S. Nadar et al. International Journal of Biological Macromolecules 2018 , 120, 2293, JA Donadelli et al. Colloids and Surfaces B: Biointerfaces 2018, 161, 654, RC Rodrigues Biotechnology Advances 2019, 37, 746, M. Bilal et al. Coordination Chemistry Reviews 2019, 388, 1):
    [0061] The method of preparing the supported biocatalyst comprises the following steps:
    [0062] 1. preparation of a magnetic nanocarrier,
    [0064] 2. surface activation of the nanocarrier,
    [0066] 3. construction of a bridging ligand and
    [0068] 4. covalent anchoring of the enzyme.
    [0070] Stages 1 and 2: Preparation of a magnetic nanocarrier and surface activation
    [0071] The magnetic nanocarrier is based on iron oxides. For its preparation, salts of Fe (II) and Fe (III) (for example, halides) are used as raw material in a molar ratio Fe (II) and Fe (III) 1: 2.
    [0073] The raw material (salts of Fe (II) and Fe (111))) is dissolved in distilled water in a reactor.
    [0075] The medium is degassed and saturated with an inert gas, preferably Argon.
    [0077] The above mixture is brought to a temperature between 60 and 80 ° C, preferably 80 ° C.
    [0079] Next, a strong base is added, preferably a base of a group 1 element. A color change of the mixture is observed to black, which indicates the formation of the mixed Fe oxides.
    [0081] The reaction lasts between 30 and 90 minutes, preferably 45 minutes for the formation of superparamagnetic nanostructures.
    [0082] Superparamantic nanostructures do not have residual magnetization when the magnetic field ceases and, furthermore, they do not emit a magnetic field as there is no external field, which prevents the nanoparticles from being added by magnetic action in the esterification / transesterification process.
    [0084] A silane with a primary amino group is then added, preferably APTES, (3-Aminopropyl) triethoxysilane).
    [0086] The silane is introduced with an excess of silane relative to Fe (II) comprised between 15 and 25% molar, preferably 20% in molar excess relative to Fe (II).
    [0088] The reaction is then maintained for a time between 1 and 3 hours, preferably 2 hours.
    [0090] After that time, the purification of the superparamagnetic nanostructures obtained with washes (for example, between 2 and 6 washes) of a buffer solution of PBS ( phosphate buffered saline) is carried out at neutral pH, preferably 7.3 and after each wash recover the superparamagnetic nanostructures by means of a magnetic decantation.
    [0092] Stages 3 and 4: Construction of the bridging ligand and covalent anchoring of the enzyme.
    [0094] In the reactor in which the superparamagnetic nanostructures are found, a dialdehyde, preferably glutaraldehyde, is added in a molar proportion equal to that of silane, (20% in molar excess with respect to Fe (II).
    [0096] A reaction occurs at room temperature, between 22 and 28 ° C. and for a time between 1 and 3 hours, preferably 2 hours.
    [0098] Analogously to the previous step, a magnetic decantation is carried out and washed with PBS.
    [0100] For the covalent anchoring of the enzyme, PBS is added again to generate reaction medium and the enzyme is added in an amount of between 3 and 6%, preferably 4% by weight relative to the amount of Fe (II) that has been added. initially (start of stage 1). The reaction is maintained for a time between 30 minutes and 2 hours, preferably 1 hour at room temperature, between 22 and 28 ° C.
    [0102] Upon completion, it is washed (eg, between 2 and 6 washes, preferably 5 times) with PBS and magnetically decanted.
    [0103] Finally, PBS is added and the suspension of the supported biocatalyst is stored at 4 ° C. The following diagram shows a particular embodiment of the procedure for obtaining the supported biocatalyst:
    [0105] APTES, H20 EtO pEt
    [0106] nh 4 oh , h 2 o , NH, FeCI, FeCI3 NPs Fe304 FesO ^
    [0107] Ar, 80 ° C, 45 min Ar, 80 ° C, 2 h
    [0109] Nanosport synthesis Construction of the bridging ligand
    [0113] PBS, pH = 7.3 Enzyme Anchor Tamb »^
    [0115] EXAMPLES OF REALIZATION
    [0117] Comparison of free biocatalyst Vs supported biocatalyst
    [0119] To compare the efficacy between a free and a supported biocatalyst, a process of glycerolysis of industrial residues (oleins) - represented in the following scheme as a fatty acid - has been carried out on a laboratory scale illustrated in the reaction scheme:
    [0121]
    [0124] The ratio of oleins to glycerol was 2: 1, and the ratio of free or supported biocatalyst was 2% by weight relative to the total weight of the reaction mixture.
    [0125] The CALB enzyme (Sigma-Aldrich) is native and for this particular embodiment it was used both in free form and anchored to the surface of the substrate.
    [0127] The glycerolysis procedure has been carried out in water and with magnetic stirring (1500rpm).
    [0129] The glycerolysis conditions are shown in the following table:
    [0134] The yield of the glycerolysis reaction was determined by an Acid-Base titration with phenolphthalein. The initial acidity of the mixture and the final acidity are measured, the difference is the moles of acids consumed, that is, the number of fatty acids that have been coupled to the glycerol.
    [0136] Assay of recovery and reuse of the supported biocatalyst
    [0138] Below is a table with the yields in the glycerolysis procedure (shown above) of the supported biocatalyst (NPs Fe 3 O 4 -CALB) after carrying out the recovery and reuse tests:
    [0140]
    [0143] The catalyst cleaning protocol before each reuse was 4 washes of 10 mL with a Triton X solution (10% v / v) at buffered pH (PBS, pH = 7.3), and subsequently 4 washes of 10 mL with a PBS buffer solution pH = 7.3. magnetic decantation of the biocatalyst, to be reused in the next glycerolysis reaction cycle.
    权利要求:
    Claims (22)
    [1]
    1. Process for the esterification and / or transesterification of oleins characterized in that it comprises carrying out the esterification and / or transesterification with a biocatalyst supported on a magnetic nano-support covalently anchored thereto.
    [2]
    2. Process according to claim 1, in which the esterification and / or transesterification process is a glycerolysis.
    [3]
    3. Process according to claim 1 or 2, further comprising optimizing the pH of the oleins at a pH of between 5.5 and 6.5.
    [4]
    4. The method according to claim 2, comprising:
    - arrange the oleins with a pH between 5.5 and 6.5 and with a water content of less than 2% and add:
    - first, the alkyl alcohols and / or polyalcohols, preferably glycerol, and
    - second, the supported biocatalyst,
    and carrying out the esterification / transesterification reaction.
    [5]
    5. Process according to the preceding claim, in which the reaction is carried out at a temperature between 50 and 70 ° C, preferably 65 ° C.
    [6]
    Process according to one of Claims 4 or 5, in which the amount of supported biocatalyst is between 0.5% and 2% of the sum of the mass of alkyl alcohols and / or polyalcohols and oleins.
    [7]
    Process according to one of claims 4 to 6, in which the amount of supported biocatalyst is in the range of 0.2-5% of the total of the reaction mixture.
    [8]
    8. Process according to one of claims 4 to 7, in which the biocatalyst is a lipase and more preferably CALB lipase.
    [9]
    Process according to one of claims 4 to 8, in which the ratio between oleins and alkyl alcohols and / or polyalcohols is 1.5: 1 to 4: 1, preferably 1.5: 1 to 2.5: 1 .
    [10]
    Process according to one of Claims 4 to 9, in which the reaction proceeds under vacuum at a pressure within the range of 0-50 mbar, preferably 0 mbar.
    [11]
    Process according to one of Claims 4 to 10, in which the reaction is carried out at a stirring of between 100 and 1000 rpm, preferably 700 rpm.
    [12]
    12. Process according to one of claims 4 to 11, in which the reaction is carried out in a time comprised between 15-30hr, preferably 24hr.
    [13]
    Process according to one of Claims 1 to 12, in which the magnetic support are superparamagnetic Fe nanostructures.
    [14]
    14. Method of preparing a supported biocatalyst to carry out the process defined in any of claims 1 to 13, comprising the following steps:
    1. preparation of a magnetic nanocarrier,
    2. surface activation of the nanocarrier,
    3. construction of a bridging ligand and
    4. covalent anchoring of the enzyme.
    [15]
    Method according to the preceding claim, in which the magnetic nanosupport are supermagnetic nanostructures based on iron oxides from salts of Fe (II) and Fe (III) as raw material in a 1: 2 molar ratio respectively.
    [16]
    16. Method according to the preceding claim, in which the raw material is dissolved in distilled water inside a reactor.
    [17]
    17. Method according to the preceding claim, in which the medium is degassed and saturated with an inert gas, preferably Argon.
    [18]
    18. Method according to one of claims 14 to 17, in which the mixing is carried out at a temperature of between 60 and 80 ° C, preferably 80 ° C.
    [19]
    19. Method according to one of claims 14 to 17, comprising adding a silane with a primary amino group, preferably APTES.
    [20]
    Method according to the preceding claim, in which the silane is introduced with a 20% excess with respect to the Fe (II).
    [21]
    21. Method according to one of claims 14 to 20, which comprises adding a dialdehyde, preferably in a molar ratio equal to that of the silane.
    [22]
    Method according to one of claims 14 to 21, comprising adding PBS to generate reaction medium and adding the enzyme in an amount of between 3 and 6%, preferably 4% by weight relative to the amount of Fe (II).
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    同族专利:
    公开号 | 公开日
    ES2804057B2|2021-07-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2695327A1|2017-06-28|2019-01-03|Consejo Superior Investigacion|SYNTHESIS OF BIODIESEL CATALYZED BY AN IMMOBILIZED ENZYMATIC CRUDE ON MAGNETIC PARTICLES |
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