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    • 专利摘要:
    A PROCESS FOR THE ENZYMATIC SYNTHESIS OF ALKY FATTY ACID ESTERS. A batch or continuous enzymatic process for the production of fatty acid alkyl esters for use in the biofuel, food and detergent industry, and a system for the same, are described. The process uses enzymes immobilized on a hydrophobic resin mixed with a source of fatty acid and an alcohol or alcohol donor in the presence of an aqueous alkaline or slightly alkaline buffer, or in the presence of a water solution. The production process for fatty acid alkyl esters is carried out by transesterification or esterification simultaneously or sequentially. Biocatalyst activity is maintained without significant activity losses in multiple uses and also prevents accumulation and prevents the accumulation of glycerol and water by-products or other hydrophilic compounds in the biocatalyst.
    公开号:BR112012022006B1
    申请号:R112012022006-3
    申请日:2011-02-02
    公开日:2021-02-23
    发明作者:Basheer Sobhi；Haj Maisa；Mohsen Usama；Shehadeh Doaa；Hindawi Ahmad；Masoud Emad
    申请人:Trans Bio-Diesel Ltd；
    IPC主号:
    专利说明:

    Field of the Invention
    An enzymatic process for the production of alkyl fatty acid esters for use in the biofuels, food and detergent industries is described. In this process, a source of fatty acid and an alcohol or alcohol donor are reacted in the presence of enzymes immobilized on a hydrophobic resin, in the presence of an aqueous alkaline buffer or water. The described process can be operated in batches or continuously using a continuous agitated tank or stuffed bed column reactors. Fundamentals of Invention
    Enzyme immobilization has been described by a vast number of techniques basically aiming at reducing the enzyme cost contribution to the total enzyme process; facilitating the recovery of enzymes from products; and the permission for continuous operation of the process.
    Immobilization techniques are generally divided according to the following: 1. Physical adsorption of enzymes to solid supports, such as silica and insoluble polymers. 2. Adsorption in ion exchange resins. 20 - 3. ~ Effective binding of enzymes to a solid support material, such as epoxidized inorganic or polymeric supports. 4. Trapping enzymes in a growing polymer. 5. Confirmation of enzymes in a membrane reactor or in semipermeable gels. 25 6. Crystals of cross-linked (CLECS’s) or aggregated (CLEAS’s) enzymes.
    All of the enzyme immobilization procedures mentioned above are comprised of the following steps: 1. Dissolve the enzyme in an appropriate buffer system in relation to pH, temperature, type of buffer salts and ionic strength. 2. Adding the solid support to the enzyme solution and mixing for some time until the enzyme molecules are immobilized on the solid support. 3. Filtration of the solid support containing the immobilized enzyme. 4. Wash the support with an appropriate buffer to remove loosely attached enzyme molecules and then dry the solid support.
    Interfacial enzymes, mainly lipases, have been immobilized following the techniques mentioned above. This offers preparations of immobilized enzymes having low synthetic activity and / or short operational half-life. In an attempt to increase the synthetic activity and stability of immobilized lipases and other interfacial enzymes, different activation methods have been applied. These methods include: 5 1. Linking groups of functional surface enzymes with hydrophobic residues, such as fatty acids or polyethylene glycol. 2. Coating the surface of enzymes with surfactants, such as polyol fatty acid esters. 3. Contact of enzymes with hydrophobic supports, typically polypropylene, which have been pre-treated with hydrophilic solvents, such as ethanol or isopropanol.
    None of the methods mentioned above produced satisfactory results in relation to stabilization and compensators in terms of the cost of immobilized interfacial enzymes, in order to perform reverse enzymatic conversions in industrial quantities. Also, it has been reported that most enzymes, when immobilized according to the procedures mentioned above, lose a significant portion of their synthetic activity or do not exhibit all of their activity performance due to certain restrictions imposed by the immobilization procedure, or due to the presence certain enzyme inhibitors in the reaction medium. 20 ~ Another major disadvantage of lipases-and phospholipases is their low tolerance to hydrophilic substrates, particularly short-chain alcohols and short-chain fatty acids (below C4). It has been observed in many research studies that short-chain alcohols and short-chain fatty acids, such as methanol and acetic acid, respectively, are responsible for detaching essential water molecules from the quaternary structure of those enzymes, leading to their denaturation and consequently to the loss of its catalytic activity. This disadvantage has prohibited the use of lipases in the production of commercial quantities of "biodiesel" of methyl esters of fatty acids using oil triglycerides and methanol as substrates.
    An additional disadvantage of using immobilized lipases for transesterification / esterification of a fatty acid source with a free alcohol is the accumulation of by-products of glycerol formed and water in the biocatalyst and, therefore, the prohibition of free access of the substrates to the active site of the immobilized enzyme. Such biocatalysts generally lose their catalytic performance after a few cycles when the same batch of biocatalyst is used. 35 The present inventors have developed special immobilized enzyme preparations, exhibiting good stability over the persistent activity of many production cycles. Examples of such enzyme preparations are described, among others, in WO / 2008/084470, WO / 2008/139455 and W02009 / 069116.
    The conditions under which the catalytic reaction is carried out can very adversely affect the stability and efficiency of immobilized enzyme preparations. It is important to have enzyme preparations that retain stability and activity under reaction conditions. These and other objectives of the invention will become apparent as the description proceeds. Summary of the Invention
    In one embodiment, the invention relates to a process for transesterification / esterification of a fatty acid source with an alcohol, to form alkyl fatty acid esters, comprising the reaction of a fatty acid source and an alcohol or a donor of alcohol in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one immobilized lipase 15 on a hydrophobic porous support and the reaction medium contains an aqueous alkaline buffer solution.
    Said aqueous alkaline buffer may be a slightly aqueous alkaline buffer. Said aqueous alkaline buffer solution can be contained in the reaction mixture in an amount of up to 5% by weight of the fatty acid source. The aqueous buffer solution may have a pH of -7-to about 11, for example, any one of 7-8.5, 7-9, 7-9.5, 7-10 and 7-11. The pKa of the slightly alkaline reagent supplemented comprising the buffer solution is greater than or equal to the pKa of the acids comprising the fatty acid source.
    In another embodiment, the invention relates to a process for transesterification / esterification of a fatty acid source with an alcohol, to form alkyl fatty acid esters, comprising the reaction of a fatty acid source and an alcohol in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support and the reaction medium contains water. The water is in the form of a water solution with a pH of 3 to 11. The reaction medium can contain the water or water solution in 5% by weight of the fatty acid source.
    In all embodiments and aspects of the invention, the alcohol can be a short-chain alcohol, for example, CI-CÔ alkyl alcohol, more specifically C1-C4 alkyl alcohol, particularly methanol or ethanol. Where said alcohol is methanol, the resulting 35 said fatty acid esters are fatty acid methyl esters (MEAG - Biodiesel). Alcohol can also be a medium-chain fatty alcohol (Cé-
    Cio) or long chain fatty alcohols (C12-C22). The alcohol donor can be a monoalkyl ester or a dialkyl carbonate, such as dimethyl carbonate or diethyl carbonate.
    In all embodiments and aspects of the invention, said immobilized lipase is capable of catalyzing the esterification of free fatty acids to produce alkyl esters of fatty acid and water as a by-product, and the transesterification of triglycerides and partial glycerides to produce alkyl esters of acid fat and glycerol as a by-product.
    In all embodiments and aspects of the invention relating to the use of an alkaline buffer 10 or alkaline solution, the amount of said buffer or alkaline solution in the reaction medium is from 0.001 to 5% by weight of the fatty acid source.
    In all embodiments and aspects of the invention, the at least one lipase may be a lipase derived from any of Rhizomucor miehei, Pseudomonas sp., Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium 15 camembertii, Alcaligenes sp. , Acromobacter sp., Burkholderia sp., Thermomyces lanuginosa, Chromobacterium viscosum, Candida antarctica B, Candida rugosa, Candida antarctica A, papaya seeds and pancreatin. Can the lipase preparation comprise at least two lipases which can each be separately immobilized on a hydrophobic support or co-immobilized on the same hydrophobic support The Teferidas lipases -can- have the same or different region specificity. Said lipases are capable of catalyzing simultaneously or consecutively the esterification of free fatty acids to produce alkyl esters of fatty acid and water as a by-product, and the transesterification of glycerides and partial glycerides to produce alkyl esters of fatty acid and glycerol with a by-product.
    In all embodiments and aspects of the invention, said support can be any one of the support based on hydrophobic aliphatic polymer and the support based on hydrophobic aromatic polymer. Said hydrophobic polymer support can be comprised of straight or branched organic chains. Said support may comprise macro-crosslinked organic polymer or copolymer chains. Said support can be porous or non-porous inorganic support, which can be hydrophobic or is coated with hydrophobic organic material. Said hydrophobic organic material can be a linear, branched or functionalized hydrophobic organic chain.
    In all embodiments and aspects of the invention in which an alkaline buffer solution 35 is used, said aqueous alkaline buffer solution can be a solution of an inorganic alkaline salt or an organic base. Said alkaline buffer solution can be a solution of any one of an alkali metal hydroxide, carbonate, bicarbonate, phosphate, sulfate, acetate and citrate, a primary, secondary and tertiary amine and any mixture thereof. In specific embodiments, said alkaline buffer solution may be a solution of a weak base selected from sodium or potassium bicarbonates and 5 carbonates. In some specific embodiments of the process of the invention, the alkaline buffer solution can be added to the fatty acid source in a premix stage or directly to the reaction medium.
    In all embodiments and aspects of the invention in which an alkaline buffer solution is used, the content of said alkaline buffer solution in the transesterification / esterification reaction medium can be in the range of 0.001-5% by weight of the oil raw material , for example, 1-2% by weight of the raw material.
    In some embodiments of the invention, the fatty acid source can first be mixed with the alkaline buffer solution or water or water solution and the mixture can then be treated with said immobilized lipase preparation, followed by the addition of the said alcohol and allowing the reaction to proceed under suitable conditions until said fatty acid source is converted to fatty acid esters.
    In all embodiments and aspects of the invention, said source of fatty acid may be one of vegetable oil, vegetable fat, algae oil, fish oil, waste oil and any mixtures of the same same source. de-fatty acid_can comprise free fatty acids, mono-, di- and triglycerides, their mixtures in any proportion, in the absence or presence of other minority fatty acid derivatives, such as phospholipids and sterol esters. The fatty acid source can be unrefined, refined, bleached, deodorized or any of its combinations.
    In all embodiments and aspects of the invention, the reaction can be carried out at a temperature between 10 ° C and 100 ° C, specifically between 25-30 ° C.
    In all embodiments and aspects of the invention, said source of fatty acid can be premixed with said alcohol or alcohol donor and with said water or buffer solution in a pre-reaction preparation vessel to form an emulsion which can then be fed together with said lipase preparation immobilized in a transesterification / esterification reaction vessel.
    In all embodiments and aspects of the invention, said lipase can be used in filled bed column reactors operating in batch or continuous modes.
    According to another aspect of the invention, a system is provided for the transesterification / esterification of a fatty acid with an alcohol, to form alkyl fatty acid esters, comprising: a reaction vessel configured to react a reaction medium including an acid fatty and at least one of an alcohol and an alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support and the reaction medium contains at least one among an aqueous alkaline buffer solution and water. io The system may comprise one or more of the following characteristics, in any desired combination or permutation: A. The reaction vessel may comprise the preparation of immobilized lipase, at least during the operation of said system for the production of said alkyl esters of acid fatty. B. In addition or alternatively to characteristic A, the reaction vessel may comprise the fatty acid and at least one of an alcohol and an alcohol donor, at least during the operation of said system for the production of said alkyl esters of acid fatty. C. In addition or alternatively to characteristics A or B, said reaction medium comprises a mixture, said-system- additionally comprising a pre-reaction vessel in selective fluid communication with said reaction vessel, said pre-reaction vessel -reaction being configured to premix at least the fatty acid and at least one of an alcohol and an alcohol donor to form said mixture, and to selectively deliver said mixture to said reaction vessel for at least during the operation of said system for the production of the fatty acid alkyl esters. The system can optionally further comprise a fatty acid source in selective fluid communication with said pre-reaction vessel and configured to selectively deliver the fatty acid to said pre-reaction vessel at least during said operation of said system, and an alcohol source in selective fluid communication with said pre-reaction container and configured to selectively deliver at least one of an alcohol and an alcohol donor to said pre-reaction container at least during said operation of said system . The system may optionally further comprise a buffer source in selective fluid communication 35 with said pre-reaction vessel and configured to selectively deliver at least one of an aqueous alkaline buffer solution and water to said pre-reaction vessel to be included in said mixture at least during said operation of said system. D. In addition or alternatively to characteristics A to C, the system can be configured to selectively deliver one or more of the fatty acid and / or 5 or at least one of an alcohol and an alcohol donor and / or at least one of an aqueous alkaline buffer solution and water for said pre-reaction vessel each in a continuous manner or in discrete batches at least during said operation of said system. E. In addition or alternatively to characteristics A to D, the pre-reaction vessel can be configured to selectively deliver said mixture to said reaction vessel in a continuous manner and / or in discrete batches at least during said operation of said system. F. In addition or alternatively to characteristics A to E, the system can be configured to selectively and directly deliver to said reaction vessel at least one of the fatty acid; o at least one of an alcohol and an alcohol donor; and at least one of an aqueous alkaline buffer solution and water. G. In addition or alternatively to characteristics A to F, said reaction vessel comprises a thermal regulation system configured for 20 reaction materials in said reaction vessel within a selected temperature range. H. In addition or alternatively to characteristics A to G, the system can optionally further comprise a holding arrangement configured to retain the immobilized lipase preparation within said reaction vessel 25 at least during the operation of said system. I.Additionally or alternatively to characteristics A to H, the system additionally comprises a product separation vessel in selective fluid communication with said reaction vessel, said system being configured to selectively deliver a reaction mixture including reaction products from the 30 said reaction vessel for said product separation vessel, and wherein said product separation vessel is configured to selectively separate a production of alkyl fatty acid esters from the reaction mixture delivered thereto. For example, the product separation container can be one of a centrifugal and gravity separation system. 35 J. In addition or alternatively to characteristics A to I, the reaction vessel is configured to selectively deliver said reaction mixture to said product separation vessel in a continuous manner and / or in discrete batches at least during said operation of that system. K. In addition or alternatively to characteristics I to J, the system is configured to selectively deliver said production of alkyl fatty acid esters from said product separation vessel. For example, the system is configured to selectively deliver said production of alkyl fatty acid esters from said product separation container in a continuous and / or discrete batch manner. L. In addition or alternatively to characteristics A to K, system 10 is configured to increase said production of fatty acid alkyl esters from the reaction mixture delivered to said product separation vessel. In a system configuration having this characteristic, the system is configured to selectively redirect said fatty acid alkyl ester production to said reaction vessel to additionally increase said production of the 15 fatty acid alkyl esters from the reaction mixture. subsequently delivered to said product separation container. In another configuration of the system having this characteristic, the system is configured to selectively redirect said production of alkyl fatty acid esters to an auxiliary reactor module, wherein said auxiliary reactor module comprises an auxiliary reactor container and an auxiliary reactor container. auxiliary product separation, wherein said additionally-increased production of alkyl fatty acid esters is selectively delivered subsequently through said auxiliary product separation vessel. Brief Description of the Figures
    In order to understand the invention and see how it can be carried out in practice, the modalities will now be described, by means of a non-limiting example only, with reference to the attached figures, in which: Figure 1: The lipase transesterification activity of Thermomyces lanuginosa (TL) immobilized on Amberlite XAD 1600 (Amb. XAD 1600) as a hydrophobic resin and on Duolite D568 (Duo D568) as a hydrophilic resin, and lipase of 30 Pseudomonas sp. (PS) immobilized on Sepabeads SP70 (SB SP70) as a hydrophobic resin and on porous silica (Sil.) As a hydrophilic resin. Abbreviations: Conv. - conversion; Cic. - Cycle Figure 2: The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction in different levels of 0.1 l sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalyst was a lipase derived from Thermomyces lanuginosa immobilized on a porous and hydrophobic polystyrene-divinylbenzene based resin. Abbreviations: Conv. - conversion; Cie. - cycle Figure 3: The conversion of soybean oil into biodiesel and glycerol after 6 hours of reaction at different levels of 0.1 M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalyst was a lipase derived from Pseudomonas sp. immobilized on a porous and hydrophobic polystyrene-divinylbenzene based resin. Abbreviations: Conv. - conversion; Cic. - cycle 10 Figure 4: The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction without water and in different levels of water using the same batch of biocatalyst in multiple batch experiments. The biocatalyst was a lipase derived from Thermomyces lanuginosa immobilized on a resin based on porous and hydrophobic polystyrene-divinylbenzene. 15 Abbreviations: Conv. - conversion; Cic. - cycle; AD - distilled water Figure 5: The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction in different water levels using the same batch of biocatalyst in multiple batch experiments. The biocatalyst was a lipase derived from Pseudomonas sp. immobilized on a resin based on porous and hydrophobic polystyrene-divinylbenzene. Abbreviations: Conv. - conversion; Cic. - cycle; AD - distilled water Figure 6: The conversion of a mixture of AGLs and soy oil into biodiesel, and glycerol and water by-products after 4 hours of esterification / transesterification in different levels of O, 1M sodium bicarbonate solution using the same batch of 25 biocatalyst in multiple batch experiments. The biocatalyst was a lipase derived from Pseudomonas sp. immobilized on a porous and hydrophobic polystyrene-divinylbenzene based resin. Abbreviations: Conv. - conversion; Cic. - cycle; AD - distilled water Figure 7: The esterification of hydrolyzed soy oil into biodiesel and water after 4 30 hours of reaction in the presence of 2% O, 1M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments . The biocatalyst was a lipase derived from Pseudomonas sp. immobilized on a porous and hydrophobic polystyrene-divinylbenzene based resin. Abbreviations: Id. Ac. - acidity level; Cic. - cycle 35 Figure 8: The transesterification of fish oil with ethanol after 6 hours of reaction in the presence of 1% by weight of 0.1 M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalysts were lipases derived from Thermomyces lanuginosa (Lip. TL) and Pseudomonas sp. (Lip. PS) immobilized on Amberlite XAD 1600. Abbreviations: Conv. - conversion; Cic. - cycle 5 Figure 9: Transesterification of Tallow fat with ethanol after 6 hours of reaction in the presence of 2% by weight of 0.1 M sodium bicarbonate solution using the same batch of biocatalyst in multiple batch experiments. The biocatalysts were lipases from Thermomyces lanuginose, Pseudomonas sp. (Lip. PS; Lip. TL) immobilized on Amberlite XAD 1600. 10 Abbreviations: Conv. - conversion; Cic. - cycle Figure 10: The treatment of the transesterification / esterification reaction medium obtained after 4 hours containing an AGL value of 7 mg KOH / lg using Pseudomonas sp. or Thermomyces lanuginosa immobilized on porous hydrophobic resins with Candida Antarctica immobilized on a porous hydrophobic resin. 15 Abbreviations: Id. Ac. - acidity level; Cic. - cycle Fig. 11: schematically illustrates a first modality of a system for the production of alkyl fatty acid esters according to an aspect of the invention. Fig. 12: schematically illustrates a second embodiment of a system for the production of alkyl fatty acid esters according to an aspect of the invention. Detailed Description of the Invention
    In the search for improvements in enzymatically catalyzed industrial processes, particularly processes for the transesterification / esterification of a fatty acid source with an alcohol in the presence of immobilized lipase / s, the present inventors have developed specific conditions under which the stability of the lipase / s 25 immobilized / s is preserved throughout production cycle counts.
    In one embodiment of the invention, the invention relates to a process for the preparation of alkyl fatty acid esters, specifically short-chain alkyl esters of fatty acids, such as methyl and ethyl fatty acid esters (biodiesel) in a micro system - solvent-free alkaline water. In specific embodiments, the alkaline micro-aqueous system 30 is a slightly alkaline micro-aqueous system. The process comprises providing a fatty acid source and reacting it with a free alcohol or an alcohol donor, in the presence of an immobilized lipase preparation, under said alkaline or slightly alkaline conditions. Without sticking to the theory, pretreating the fatty acid source with an alkaline buffer solution would result in the neutralization of acids that can have an inhibitory effect on the enzyme. The amount of alcohol required to complete the reaction until 100% conversion can be added step by step or in a batch. In addition, the alcohol may be short-chain alcohol, for example, methanol or ethanol. Other alcohol donors can be used in the reaction with the fatty acid source in the presence of a hydrolase and allowing the reaction to proceed under suitable conditions, until the said fatty acid source is converted into alkyl fatty acid esters, specifically, fatty acid methyl esters (MEAG) or fatty acid ethyl esters, wherein said hydrolase preparation comprises one or more lipases, immobilized separately or together on a support based on a suitable macro-cross-linked porous hydrophobic polymer. 10 In an additional modality, the transesterification / esterification reaction between the fatty acid source and the alcohol or alcohol donor is carried out in an aqueous microenvironment, with the addition of water to the reaction mixture. In specific embodiments, water can be added at 0.0001 to 5% by weight of the fatty acid source. By water as used here, it means pure or distilled water, and also “water solutions”, which can be, but are not limited to, tap water, sea water or water from any other resource or reservoir. natural water, desalinated water, purified or chemically or enzymatically treated water, and any other aqueous solutions. The pH of the reaction system or water solution can vary, or it can be, for example, about 3-11, for example, 4-10, 5-10, 5-9, 6-10, 6-9 or 7-9. The process of the invention can be carried out while continuously removing the formed glycerol and any excess water from the reaction mixture. The conversion of the acyl groups of fatty acids or free fatty acids comprised in said fatty acid source to alkyl fatty acid, specifically methyl esters can be monitored at various points during the reaction. The reaction medium can be removed by 25 suitable means at any desired time point during the reaction, thereby stopping the reaction, and the fatty acid methyl esters formed and optionally the glycerol formed are isolated from the reaction medium. The reaction can be specifically stopped when the conversion of the acyl groups of fatty acid and free fatty acids comprised in the said fatty acid source into fatty acid methyl esters reaches at least 30 70%, for example, at least 85% or at least 90% .
    The reaction system can be similar to the one described in co-pending with W02009 / 069116. For example, the production system may use an agitated tank reactor with a sintered glass or stainless steel bottom filter that retains the biocatalyst the reactor, however, allows the reaction medium to permeate through the reactor. Such a reactor configuration allows by-products, specifically glycerol and water, which are self-absorbed from the immobilized enzyme, to sink to the bottom of the reactor, and permeate through the filter. The result is the continuous removal of the desorbed glycerol formed and also of excess water, out of the reaction medium, leading to the reaction moving towards synthesis, thereby achieving conversions above 98%. The biocatalyst used in this reactor can be comprised of lipases of 5 single or multiple types, in consideration of their positional specificity, as well as their origin, as described here. Alternatively, two consecutive agitated tank reactors with a fuel filter can be used. A deposition tank or centrifuge can be used between the two reactors. The first reactor may contain an immobilized biocatalyst comprised of single or multiple types of lipases. The function of the deposition tank or centrifuge between both reactors is to remove the formed glycerol and excess water from the reaction medium, leading to an increase in the conversion of crude materials in their corresponding fatty acid alkyl esters to above 98% in the second reactor in reasonable reaction time. Some specific reaction systems and methods are described below. 15 The terms "reaction mixture", "reaction system" and "reaction medium" can be used here synonymously.
    The use of lipases immobilized on hydrophobic resins in the presence of alkaline buffer solution or water, as in the modalities of the process of the invention, ensures the high stability of the enzyme and also prevents the accumulation of hydrophilic substances, such as water and the glycerol by-product formed , in the biocatalyst. Specific procedures - of the process of the invention, 0.001-5% of alkaline or slightly alkaline buffer solution is used, for example, 0.01-5%, 0.05-5%, 0.1-5%, 0.5 -5%, such as 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%. In specific embodiments of the process of the invention in which water is used, water is used at levels of 0.0001-5% water, for example, 0.001-5%, 0.01-5%, 0.05-5%, 0.1- 5%, 0.5-5%, such as 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 0.5%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4 %, 4.5% or 5%. As mentioned, when the alkaline solution is used, it is possible to neutralize acids typically present in the fatty acid source or to produce due to side reactions. The continuous active removal of these 30 by-products can even increase the efficiency of the process. Isolated glycerol can be used industrially.
    The fatty acid source used in the process of the invention can comprise at least one of soy oil, canola oil, seaweed oil, rapeseed oil, olive oil, castor oil, palm oil, sunflower oil, peanuts, cottonseed oil, jatropha oil, crude corn oil, fish oil, animal-derived fat, residual cooking oil, brown grease, oil triglycerides derived from non-edible plant sources, partial glycerides and fatty acids free derivatives of these oils or any mixture of at least two of them, in any desired proportion.
    In all of the processes of the invention, the alkyl short-fatty acid esters formed by the reaction are specifically methyl, ethyl, isopropyl or butyl fatty acid esters (biodiesel). Other medium-chain fatty alcohols (Cg-C10) and long-chain fatty alcohols (C12-C22) can also be used in the production process of that invention. These longer alcohols may be specifically suitable in the production of waxes, for example, for cosmetic products. 10 The lipases can be lipases derived from Thermomyces lanuginosis, Rhizomucor miehei, Mucor miehei, Pseudomonas sp., Rhizopus sp., Mucor javanicus, Penicillium roqueforti, Aspergillus niger, Chromobacterium viscosum, Acromobactertica, Candida sp. antarctica B, Candida rugosa, Alcaligenes sp., Penicillium camembertii, papaya seeds and pancreatin, but are not limited to them.
    The lipases can be immobilized together on a suitable support, specifically a support based on hydrophobic aliphatic polymer or a hydrophobic aromatic polymeric support. Each of said lipases can be immobilized on a suitable support, wherein the supports on which said lipases are immobilized are identical or different. The lipases employed can be regiospecific to their substrate or random. When more than one lipase is used, the lipases can be immobilized on the same support or on different hydrophobic supports. Lipases co-immobilized on the same support may exhibit identical or different substrate selectivities or region specificities to their substrates. 25 Lipases can be regiospecific (or site specific), each used alone or in combination with lipases of the same or different site specificity. When referring to sn-1, sn-2- or sn-3 positions, these are positions on the glycerol backbone of the various glycerides. Thus, the lipases used in the process of the invention may have selectivity towards the sn-2 position higher than that of random lipases, that is, their favor catalyzing the reaction between alcohol and the alcohol donor with the fatty acyl group of position sn-2, while random lipases exhibit the same transesterification activity for fatty acyl groups in all three positions on the glycerol backbone. Some lipases exhibit only positional activity at the sn-2 position, specifically under specific conditions 35 determined by substrates, products, etc. Other lipases used in the process of the invention are specific positional sn-1,3. They can be used alone or in conjunction with a random lipase, specifically a lipase that has an affinity for partial glycerides, and optionally a third lipase with a high affinity for the sn-2 position.
    The support is specifically a macro-reticular and porous hydrophobic support, 5 which can be organic or inorganic. Examples of supports are porous inorganic supports, such as, but not limited to, hydrophobized silica and / or alumina based supports, and hydrophobic organic supports, such as, but not limited to, polymer or polymer based supports. The supports can optionally contain functional groups selected from epoxy and / or aldehyde groups or ionic groups. The insoluble support used in the processes of the invention is specifically a support based on crosslinked and porous hydrophobic aliphatic or aromatic polymer, such as AmberliteR XAD 1600 and SepabeadsR SP70, both comprised of porous micro-reticular resin prepared from divinylbenzene or from a mixture of divinylbenzene and polystyrene, AmberliteR XAD 7HP comprised of micro-reticular aliphatic acrylic polymer 15, and porous aliphatic polymer, such as a porous polypropylene (AccurelR).
    The support may be a crosslinked hydrophobic polymer comprised of divinylbenzene, or a mixture of divinylbenzene and styrene, and hydrophobic aliphatic polymer comprised of aliphatic acrylic polymers or polyalkylene, such as 2Õ specific propylene polystyrene porous matrices, of such a large size in 25 porous range in the porous range -1000 Â, and more specifically in the 80-200 Â range. The support can also be granular or powdered porous hydrophobic silica or other inorganic oxides. The support can also be granular or powdered porous hydrophobized silica and other inorganic oxides. In specific modalities, the surface area of the support resins is 25 greater than 100m2 / g.
    The amount of the alkaline or slightly alkaline aqueous solution to be supplemented in the lipase catalyzed transesterification / esterification reaction between the fatty acid source and the alcohol is generally below 5% by weight of the reaction medium. This alkaline solution is prepared, for example, from an inorganic alkaline base or salt or from an organic base. Inorganic bases and salts are, for example, alkali metal hydroxides, carbonates, bicarbonates, phosphates, sulphates, acetates and citrates. The organic bases can be, for example, primary, secondary or tertiary amines. Mixtures of these alkaline agents are also contemplated. In the process according to the invention, the pH of the immobilized enzyme microenvironment is maintained at alkaline or slightly alkaline values. While the addition of distilled water to the reaction system increases the performance of immobilized lipases on the hydrophobic support (resins), as shown in Figures 4 and 5, the addition of several alkaline buffers, with different pH values depending on the type of base used , resulted in additional stabilization of immobilized lipases on hydrophobic supports (resins), as shown, for example, in Figures 2 and 3. The carbonate and 5 bicarbonate buffers are examples of light bases that are efficient in increasing the stability of immobilized lipases in hydrophobic supports. Other suitable bases are described here. Generally, the pKa of the alkaline or slightly alkaline reagent comprising the buffer solution is equal to or greater than the pKa of acids comprising the fatty acid source. The alkaline solution as used here is generally a solution with a pH 10 of 7 to about 11, for example, 7-8.5, 7-9, 7-9.5, 7-10 or 7-11. Generally, the amount of alkaline or slightly alkaline aqueous solution used is expressed in percentages by weight (% by weight) based on the amount of oil used in the reaction.
    The use of lipases immobilized on supports based on porous hydrophobic polymers (resins) in the presence of an alkaline or slightly alkaline solution, for example, in an amount of 0.01-5% by weight, 0.05-5% by weight , 0.05-4% by weight, 1-5% by weight, or 1-4% by weight, results in the stabilization of the activity of biocatalysts in the transesterification / esterification reactions between the fatty acid source and alcohol. This is also shown in the following Examples.
    The fatty acid source is at least one of triglycerides, 20 partial glycerides, free fatty acids, phospholipids, fatty acid esters and amides or a mixture comprised of at least two of the said sources.
    The production of alkyl fatty acid esters is carried out by transesterification or esterification, simultaneously or sequentially. Under such a reaction system, biocatalyst activity is maintained without significant activity losses in multiple uses and also prevents the accumulation of glycerol and water by-products or other hydrophilic compounds in the biocatalyst.
    The invention provides processes employing specific immobilized interfacial enzymes that retain high activity and stability over many production cycles. Specifically, the preparations of lipases and phospholipases are used in 30 transesterification / esterification reactions. These reactions can be used in the production of food items, cosmetics and biofuels ("biodiesel"). Of particular interest, these enzymes can be used for the synthesis of short chain fatty acid alkyl esters for use as "biodiesel".
    The present invention employed stable immobilized interfacial enzymes, highly tolerant to short chain alcohols, such as methanol, ethanol and glycerol, as well as short chain fatty acids, such as acetic acid. The use of these enzyme preparations also prevents the accumulation of hydrophilic substances, particularly glycerol and water, in the immobilized biocatalyst.
    In one embodiment of the invention, a process is provided for simultaneous or sequential transesterification / esterification reactions of a fatty acid source 5 with an alcohol using one or more types of lipases, immobilized on a hydrophobic support (resin), in the presence of a solution aqueous alkaline or slightly alkaline, to obtain the desired product, namely, alkyl esters of fatty acid, close to completing conversions during a reasonable reaction time, typically below 5 hours. A slightly alkaline solution, for example, a 0.001M, 10%, 1M, 0.5M or 1M sodium bicarbonate solution, can be presented in the reaction system in an amount of less than about 5% by weight or about 4% by weight of the amount of oil used in the reaction.
    As shown in the Examples below, the operating life of lipases can also be extended by using hydrophobic resin support for lipase immobilization in combination with the use of an alkaline or slightly alkaline buffer solution, for example, in the 0.001-5% by weight in the transesterification / esterification reaction medium. As further shown in the Examples below, the water content of the reaction mixture can be increased regardless of the pH value. Thus, in another modality, the stability of the biocatalyst increases 20 with the increase of the water content ~ reaction system by the addition of water, for example, by 0.0001-5% by weight of the fatty acid source, any of the sub-bands specifics defined above. The results show that the addition of an alkaline solution in the range of 0.0001-5% by weight of the fatty acid source (Figures 2 and 3) or water in 0.001-4% of the fatty acid source (Figures 4 and 5) results in the maintenance of enzyme activity and stability over many reaction cycles.
    The alcohol or alcohol donor employed in the processes of the invention can be a lower alkyl alcohol, specifically Cj-Cé alkyl alcohol, more specifically C1-C4 alkyl alcohol, and particularly methanol or ethanol or the alcohol donor can be monoalkyl ester or dialkyl carbonate, such as dimethyl carbonate. An alcohol donor, such as, for example, dialkyl carbonate, can also serve as a source for the alkalinity or mild alkalinity of the reaction system.
    According to another aspect of the invention, a system is provided for the production of alkyl fatty acid esters. Referring to Fig. 11, a first embodiment of such a system, generally designated with the reference numeral 100, comprises a reactor vessel 120, a pre-reaction preparation vessel 140 and a product separation vessel 160.
    The pre-reaction preparation container 140 is configured to receive raw material materials and buffer (and / or water), to form a suitable emulsion therefrom, and to feed the prepared EP emulsion (also referred to here as emulsified raw material) for reactor container 120. In particular, such raw material materials may include AG fatty acid (eg residual cooking oil) from a 182 fatty acid source, and AL alcohol (eg methanol) from the alcohol source 184, and buffer (and / or water) TA from the buffer source / water 10 186, provided through suitable supply lines 152, 154, 156, respectively, in fluid communication with the said pre-reaction preparation container 140 via container inlets 172, 174, 176, respectively, and suitable valves (not shown).
    The pre-reaction preparation container 140 defines an internal volume VI at 15 in which the reaction mixture, including the raw material and buffer / water materials, provided through the container inlets 172, 174, 176, are mixed together by means of a suitable stirring system 142, driven by an energy source (not shown), to form the EP emulsion. The pre-reaction preparation container 140 comprises an outer casing 149 through which a suitable working fluid 20 can be circulated to maintain volume VI at a desired stationary temperature. For example, the working fluid can be oil or water, heated or cooled in a different container (not shown) and pumped through housing 149 through suitable inlet and outlet ports (not shown). In alternative variations of this embodiment, the pre-reaction preparation container 140 may comprise a system 25 of heating and / or cooling elements, for example, electrically powered heating and / or cooling elements, instead of or in addition to housing 149 .
    The reactor container 120 is configured to receive the prepared EP emulsion from the pre-reaction preparation container 140, to react the raw material materials therein, in the presence of a suitable BC biocatalyst to produce PR reaction products, and to feed the PR reaction products from the reaction mixture to the product separation vessel 160. The outlet line 148 provides selective fluid communication between the pre-reaction preparation vessel 140 and the reactor vessel 120 through suitable valves (not shown) and allows the prepared emulsion EP 35 prepared by the pre-reaction preparation container 140 to be fed to the reactor container 120 as desired.
    The reaction vessel 120 defines an internal volume V2 in which the prepared emulsion EP in the reaction mixture, provided therein via the vessel inlet 122, is reacted, and the reaction mixture can be stirred by means of a suitable stirring system. 124, driven by an energy source (not shown) to form the PR reaction products. The BC biocatalyst can comprise a suitable enzyme and is provided in the form of bundles of immobilized enzymes that remain in the reactor vessel 120 until they become inefficient or not sufficiently efficient, on which they can be removed with the new BC biocatalyst. For example, the BC biocatalyst may comprise lipase derived from Thermomyces lanuginosa 10 immobilized on a resin based on porous or hydrophobic polystyrene-divinylbenzene.
    The reactor vessel 120 comprises a thermal regulation system in the form of an outer shell 129 through which the working fluid can be circulated to maintain volume V2 at a desired stationary temperature. For example, the working fluid 15 can be oil or water, heated or cooled in a different container (not shown) and pumped through housing 129 through suitable inlet and outlet ports 123. In alternative variations of this modality, the thermal regulation comprises a system of heating and / or cooling elements, for example, electrically powered heating and / or cooling elements, instead of or in addition to housing 129;
    The lower part of the reactor container 120 comprises an outlet 127 and a suitable retention arrangement in the form of filter 125 is provided upstream of the outlet 127 configured for filtering the reaction mixture, particularly the PR reaction products before being removed from the reactor container. 120 and prevent the BC biocatalyst from being removed with the PR reaction products.
    The product separation vessel 160 is configured to separate, from the reaction products PR, the desired product P (fatty acid alkyl ester), from by-products including excess water and glycerol G. Output line 147 provides communication selective fluid between product separation vessel 160 and reactor vessel 120 via suitable valves (not shown) and allows reaction products PR to be fed to product separation vessel 160 from reactor vessel 120 as desired. In this embodiment, the product separation vessel 160 comprises a centrifugal or gravity separation system to perform the aforementioned separation, and includes a first outlet 162 35 to flow product P, and a second outlet 164 to collect excess water and glycerol G. Product P can be collected through tap 163.
    The system can thus be operated in a continuous production mode, in which the prepared emulsion EP is fed to the reactor container 120, and the desired product P is collected continuously through tap 163. The EP emulsion can be prepared and delivered continuously to the reactor vessel 120 to compensate for the volume of reagent at the same rate as reaction products PR are removed from outlet 127. Alternatively, the EP emulsion can be prepared and delivered in batches to the reactor vessel 120 to compensate for the volume of reagent in the reaction mixture at discrete intervals whenever the level of reagents in the reactor vessel 120 drops to a particular minimum level according to the continuous removal of reaction products PR through outlet 127. Of course, it is also possible to operate the system 100 to provide the desired product P in batches instead of continuously.
    Alternatively, system 100 can be operated to improve the mode of production, where product P is, instead of being immediately collected via tap 163, forwarded to reactor container 120 via a forwarding system, including line 165, the container inlet 121 and valve 166, where valve 166 can be selectively operated to bypass product P from tap 163. When forwarded to reactor container 120, product P can be further reacted in it with alcohol AL, provided via a separate line (not shown) from source 184, from a different alcohol source (not shown), or from source 184 through the pre-reaction preparation vessel, to produce greater production of product P, which again can be separated from the by-products using the product separation container 160. When the alcohol is supplied through the preparation container 140, it is first emptied of the emulsion p repaired EP, and suitable valves prevent AG fatty acids and optionally the buffer / water from being provided by the respective sources 182 and 186. Suitable pumps or gravitational feed and controllable valves can be provided to selectively transport the respective materials through the respective lines 152, 154, 156, 148, 147, 165, and a suitable controller (not shown) monitors and controls the operation of the system.
    In at least some alternative variations of the first embodiment, the pre-reaction preparation container 140 can be integral with the reactor container 120. For example, the respective internal volumes VI and V2 can be separated by a wall containing an opening arrangement corresponding to line 148. Alternatively, the respective internal volumes VI and V2 can be contiguous, but the internal volume VI is sufficiently spaced from the BC biocatalyst to provide sufficient time for the EP emulsion to form before reaching the BC biocatalyst.
    In alternative variations of the first modality, one, two or all of the fatty acid AG, alcohol AL and buffer / water TA can be supplied directly to the reactor container 120, avoiding the pre-reaction preparation container 140. For example, one or more sources of fatty acid 182, source of alcohol 184 and source of 5 buffer / water 186, can be in selective fluid communication directly with the reactor vessel 120, through appropriate supply lines (not shown) avoiding the preparation vessel of pre-reaction 140.
    It is appreciated that all components of the system 100 according to the first modality, or alternative variations thereof, are suitably made from suitable materials as known in the art, in order to allow each component to perform its respective functions in the respective conditions, including temperature, pressure, pH and so on.
    Referring to Fig. 12, a second modality of the system, designated with reference number 200, comprises all the elements and characteristics of the first modality, including alternative variations thereof, including all components numbered similarly to Fig. 11 , mutatis mutandis, with some differences. For example, system 200 also comprises: a reactor vessel 120, a pre-reaction preparation vessel 140, a product separation vessel 160, a fatty acid source 182, alcohol source 184, buffer / water source 186 , 2Õ supply lines 152, 154, 156, container inlets 172, 174, 176, stirring system 142, outer shell 149, outlet line 148, container inlet 122, stirring system 124, biocatalyst BC, outer shell 129 , input and output ports 123, output 127, filter 125, output line 147, first output 162, second output 164; as described for the first modality, mutatis mutandis. However, in the second embodiment, line 165, tap 163 and valve 166 of the first embodiment are omitted and, instead, an auxiliary reactor module 300 is operatively connected to the first outlet 162 of the product separation vessel 160 .
    The auxiliary reactor module 300 comprises an auxiliary reactor container 220 and 30 an auxiliary product separation container 260, which in this embodiment are respectively substantially similar to the reactor container 120 and the product separation container 160, mutatis mutandis. In operation, the desired product P from the product separation container 160 is sent to the auxiliary reactor container 220 via line 266, valve 267 and container inlet 221. When forwarded 35 to the auxiliary reactor container 220, the product P can be additionally reacted therewith with AL alcohol, provided via a separate line (not shown) from source 184 or a different alcohol source (not shown), to produce additional reacted PRA products. Line 249 allows PRA reacted products to be transported to the auxiliary product separation container 260, which then operates to separate higher production of product P 'from by-products. System 200 can be operated in a similar manner to system 100, mutatis mutandis.
    Described and exposed, it should be understood that this invention is not limited to the particular examples, process steps, and materials described here, since the process and material steps may vary in some way. It should also be understood that the terminology used here is used for the purpose of exposing some modalities only and is not intended to be limiting since the scope of the present invention will be limited only by the claims and equivalents thereof.
    It should be noted that, as used in that specification and in the appended claims, the singular forms "one", "one" and "o / a" include plural referents, unless the content clearly indicates otherwise.
    Throughout this specification and the claims that follow, unless the context otherwise requires, the word "understand", and variations such as "understand" and "understanding", will be understood to imply the inclusion of an affirmed or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps;
    The following Examples are representative of the techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that, while these techniques are exemplary of the preferred modalities for the practice of the invention, the one skilled in the art, in the light of the present description, will recognize that countless modifications can be made without deviating from the intended scope of the invention.
    Examples General All experiments were carried out in glass tubes of 30 ml volume with a glass filter centered on the bottom or in mechanically agitated reactors of 30 500 ml volume with a sintered glass filter on the bottom of porosity of 150-250 μm . The typical reaction medium contained a source of fatty acid, alcohol, usually methanol or ethanol on a 1: 1 molar basis in relation to the fatty acid regardless of whether it was free or attached to a glycerol backbone (for fatty acids and monoglycerides 1: 1, for diglycerides 1: 2 and triglycerides 1: 3 in favor of alcohol). The source of fatty acid 35 was pre-mixed with different amounts of alkaline buffer, in specific modalities, sodium bicarbonate. The reactions were initiated by the addition of immobilized lipase on a hydrophobic resin (10-15% by weight) and the reaction medium was mechanically stirred or stirred at 30 ° C. The amount of alcohol was also added in three steps, each one hour apart, unless otherwise indicated. The reaction conversions were followed by taking samples from the reaction medium at different time intervals and analyzing the fatty acid components. The conversion to biodiesel was calculated as 100 * of the fatty acid alkyl ester peak area / sum of all the peak areas.
    Lipase immobilization: The lipases were immobilized following standard procedures in which the lipase derived from a certain microorganism is solubilized in a 0.1 M buffer solution at a certain pH value, for example, 7.5. An organic or inorganic polymer resin was introduced into the lipase solution. The mixture was stirred at room temperature for 8 hours. Cold acetone was optionally added to the mixture in order to increase the protein enzyme precipitation on the resin. The mixture was filtered and the enzyme bundles were dried to reduce the water content to less than 15% of 5%.
    Different resins were used including hydrophobic polymer reins based on polystyrene / divinylbenzene, paraffin or any of their combinations, to obtain resins with hydrophobic characteristics. Typical hydrophobic resins used included AmberIiteR XAD 1600 (Rohm & Haas, USA) and SepabeadsR SP70 (Resindion, 20 Italy). Typical hydrophilic resins used included DuoliteR D568 (Rohm & Uaas) and porous silica gel. Lipases can be immobilized separately on a resin or different lipases are co-immobilized on the same resin.
    Example 1 The transesterification activity of the lipase derived from Thermomyces 25 lanuginosa immobilized on AmberliteR XAD 1600 as a hydrophobic resin and on DuoliteR D568 as a hydrophilic resin, and the lipase derived from Pseudomonas sp. immobilized on SepabeadsR SP70 as a hydrophobic resin and on porous silica as a hydrophilic resin.
    Reaction conditions: Refined and bleached soybean oil (20 g) containing 1% 30 wt.% 0.1M sodium bicarbonate solution. Methanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. The reaction medium containing 10% by weight of the lipase preparation was stirred at 300 rpm and 30 ° C. The results are shown in Figure 1.
    The results shown in Figure 1 show that both the lipases of 35 Thermomyces lanuginosa and Pseudomonas sp. immobilized on different resins in the presence of 1% by weight of sodium bicarbonate solution showed high transesterification activity during the first 5 cycles using the same batch of enzyme. It was observed that after the 5th batch, when the same enzyme batch was used, filtration of the reaction medium from the system became difficult due to the formation of a gel-like deposit around the bundles of both immobilized lipases on 5 hydrophilic resins, namely DuoliteR D568, and porous silica. The transesterification activity of both lipases immobilized on hydrophilic resins decreased sharply in additional consecutive batches, and became inactive after the 10th cycle. In contrast, the lipase of Pseudomonas sp. immobilized on the hydrophobic resin, SepabeadsR SP70, retained more than 80% of its initial activity after 70 cycles, while 10 that the Thermomyces lanuginosis lipase immobilized on the hydrophobic resin, AmberliteR XAD 1600, retained more than 20% of its initial activity after more than 70 cycles.
    Example 2 A. The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.
    Reaction conditions: Refined and bleached soybean oil (20 g) containing different concentrations of 0.1 l sodium bicarbonate solution. Methanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. Thermomyces lanuginosa-derived lipase immobilized on a porous and hydrophobic polystyrene-divylbenzene-based resin 20 was used (+ 0% weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 2.
    B. The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments. 25 Reaction conditions: Refined and bleached soy oil (20 g) containing different concentrations of 0.1 l sodium bicarbonate solution. Methanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. Lipase derived from Pseudomonas sp. immobilized on a resin based on porous and hydrophobic polystyrene-divinylbenzene was used (10% by weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 3.
    Figures 2 and 3 show that the amount of sodium carbonate in the reaction medium has an important role in the operational life of the lipases of Thermomyces lanuginosa and Pseudomonas sp. immobilized on hydrophobic resins. It can be seen in Figures 2 and 3 that, in the absence of an alkaline solution, both immobilized lipases 35 have drastically lost their activity after a few cycles, while the same immobilized lipases maintain their transesterification activity over multiple uses in the presence of sodium bicarbonate solution as a base in the reaction system. The results for both immobilized enzymes show that the increase in the amount of sodium bicarbonate solution in the reaction medium in the range of 0 - 4% by weight results in the decrease in enzyme loss and activity in the multiple uses of the same immobilized enzyme batch. .
    Example 3 A. The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.
    Reaction conditions: Refined and bleached soybean oil (20 g) containing 10 different concentrations of distilled water. Methanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. The lipase derived from Thermomyces lanuginosa immobilized on a resin based on porous and hydrophobic polystyrene-divinylbenzene was used (10% by weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 4. 15 B. The conversion of soy oil into biodiesel and glycerol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.
    Reaction conditions: Refined and bleached soybean oil (20 g) containing different concentrations of distilled water. Methanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. Lipase derived 20 from Pseudomonas sp. immobilized — on — a resin based on porous, hydrophobic polystyrene-divinylbenzene was used (10% by weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 5.
    Figures 4 and 5 show that the transesterification activity using the same batch of lipases from Thermomyces lanuginosa and Pseudomonas sp. immobilized 25 on hydrophobic resins in multiple experiments is also affected by the amount of water in the reaction system. It can be seen that the increase in the amount of water from nothing (zero) to 4% by weight resulted in the maintenance of transesterification activity of the highest residual biocatalyst when used in consecutive cycles. The results presented in Figures 2 to 5 evidently show that the use of light base, such as sodium bicarbonate solution, in the transesterification reactions is favorable for the maintenance of the activity of immobilized lipases on hydrophobic resins when used in consecutive cycles.
    Example 4 The conversion of a mixture of free fatty acids (AGLs) and soy oil into 35 biodiesel, and glycerol and water by-products after 4 hours of esterification / transesterification using the same batch of biocatalyst in the multiple batch experiments.
    Reaction conditions: A mixture of soy hydrolyzate and free fatty acids (50% by weight) and soy oil (50% by weight) with an initial AGL value of 72 mg KOH / lg containing different amounts of sodium bicarbonate solution of O, 1M. Methanol (4.5 ml) was added step by step in three equivalent batches, each one hour apart. Lipase derived from Pseudomonas sp. immobilized on a porous and hydrophobic polystyrene-divinylbenzene resin was used (20% by weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 6.
    Figure 6 shows that the different amount of basic solution has an important effect on the simultaneous esterification reaction of AGL present in reaction mixture 10 comprised of different proportions of soybean oil hydrolyzate and soybean oil triglycerides. It can be seen that the lipase of Pseudomonas sp. immobilized on a hydrophobic resin lost its esterification activity when no alkaline solution was added to the esterification / transesterification reaction system, while the same biocatalyst maintained its activity in consecutive cycles 15 when 1 and 2% by weight of sodium bicarbonate solutions 0.1 M were added separately to the reaction systems. The results shown in Figure 6 show that the use of lipase Pseudomonas sp. immobilized on a hydrophobic resin reduced the FFA content in the presence of 1% and 2% by weight of 0.1 lM sodium bicarbonate solution from the initial value of 72 mg KOH / lg to 8 and 6 mg KOH / lg in average, 20 respectively, and maintained this activity in 22 subsequent cycles.
    Example 5 The esterification of soybean oil hydrolyzate in biodiesel and water after 4 hours of reaction using the same batch of biocatalyst in multiple batch experiments.
    Reaction conditions: Soy hydrolysate and free fatty acids (20 g) of 25 AGL value of 150 mg KOH / lg containing 1% by weight of 0.1 l sodium bicarbonate solution. Methanol (2 ml) was added to the reaction medium in a batch. Lipase derived from Pseudomonas sp. immobilized on a resin based on porous and hydrophobic polystyrene-divinylbenzene was used (10% by weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 7. 30 Figure 7 shows that the lipase from Pseudomonas sp. immobilized on a hydrophobic resin is also able to catalyze the esterification of free fatty acids to form fatty acid methyl esters and a water by-product. The results show that the lipase preparation maintained in this esterification / transesterification activity in a medium containing 1% 0.1M sodium bicarbonate solution over more than 25 35 cycles using the same batch of biocatalyst without the observation of any significant loss of activity.
    Example 6 Transesterification of fish oil with ethanol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.
    Reaction conditions: Refined fish oil (20 g) containing 1% solution of 5, 1M sodium bicarbonate. Ethanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. Lipases derived from Thermomyces lanuginosa and Pseudomonas sp. immobilized on AmberliteR XAD 1600, were used separately (10% by weight). The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 8. 10 Figure 8 shows that both lipases derived from Thermomyces lanuginosa and
    Pseudomonas sp. immobilized on hydrophobic resins are also able to catalyze the transesterification of fish oil triglycerides with ethanol to form fatty acid ethyl esters and a glycerol by-product. The results also show that both biocatalyst preparations maintained their transesterification activity 15 in the presence of 1% by weight of sodium bicarbonate solution without significant activity losses over more than 20 cycles using the same batch of biocatalyst.
    Example 7 Transesterification of Tallow fat with ethanol after 6 hours of reaction using the same batch of biocatalyst in multiple batch experiments.
    Reaction information: Tallow fat (16 g) containing Tallow fatty acid ethyl ester (4 g) and 1% 1M potassium carbonate solution. Ethanol (2.5 ml) was added step by step in three equivalent batches, each one hour apart. Lipases derived from Thermomyces lanuginosa, Pseudomonas sp. immobilized on AmberliteR XAD 1600 (10% by weight) 25 were used separately or in combination in an equivalent proportion. The reaction medium was stirred at 300 rpm and 37 ° C. The results are shown in Figure 9.
    Figure 9 shows that both lipases derived from Thermomyces lanuginosa and Pseudomonas sp. immobilized on hydrophobic resins are able to catalyze the transesterification of tallow fat triglycerides with ethanol to form 30 fatty acid ethyl esters and a glycerol by-product. The raw material of the reaction medium was comprised of tallow fat (80%) and fatty acid ethyl esters derived from tallow fat in order to reduce the melting point of the reaction medium. The results presented in Figure 9 show that all biocatalysts retained more than 80% of their initial activity in the presence of a slightly alkaline solution, such as 1M potassium carbonate 35, when the same batch of biocatalysts were used in 100 consecutive cycles.
    Example 8 The treatment of the transesterification / esterification reaction medium obtained after 4 hours containing AGL value of 7 mg KOH / lg using lipase from Thermomyces lanuginosa or lipase Pseudomonas sp. immobilized on a porous hydrophobic resin 5 with Candida antarctica B lipase immobilized on a porous hydrophobic resin and methanol (1:10 ratio in molar base between AGL and methanol, respectively) using the same batch of biocatalyst (10% by weight) in multiple batch experiments. The reaction medium was stirred at 300 rpm and 30 ° C. The results are shown in Figure 10.
    Figure 10 shows that the transesterification reaction medium obtained after treatment with Thermomyces lanuginosa lipase or Pseudomonas sp. as described above, which typically contains AGL values of 3-7 mg KOH / lg, can be treated with Candida antarctica B lipase immobilized on hydrophilic or hydrophobic support, the results reducing the ALG value to less than 2mg KOH / lg. The immobilized lipase can maintain its activity in more than 100 cycles.
    Example 9 Transesterification / esterification of residual cooking oil containing 10% FFA with methanol to form biodiesel, water and glycerol using the first modality of the system illustrated in Figure 11.
    Reaction conditions: Residual cooking oil (1100 g) containing 2% 0.1 M sodium bicarbonate solution of methanol (140 g) was first pre-mixed in the pre-reaction preparation vessel 140 to form an emulsion, which was then introduced into the reactor vessel 120 having an internal volume of V2 of about 2 liters. The reaction mixture was mixed in reactor 120 with a lipase derived from Thermomyces lanuginosa immobilized on a porous and hydrophobic polystyrene-divinylbenzene based resin (30% by weight of oil) for 6 hours at 30 ° C. The reaction mixture was filtered through filter 125 and fed to the product separation vessel 160. Glycerol and excess water were removed from the reaction mixture in the product separation vessel 160. The upper phase containing the methyl 30 esters of fatty acid and unreacted glycerides were re-introduced into reactor vessel 120 via redirection line 165, and stirring of reactor vessel 120 was continued after adding methanol (110 g) to the reaction medium in reactor vessel 120. A conversion to methyl ester after 2 hours was 98%. An emulsified reaction medium (prepared emulsion) containing residual cooking oil (83% by weight), 35 methanol (15%) and 0.1M sodium bicarbonate solution (2%) was continuously fed to reactor vessel 120 in a flow rate of about 30ml / min. The conversion to fatty acid methyl esters was maintained for more than 3 months without significant loss of activity when using the same lot of biocatalyst derived from Thermomyces lanuginosa lipase immobilized on a macroporous hydrophobic resin.
    权利要求:
    Claims (15)
    [0001]
    1. Process characterized by being selected from: (A) a process for the transesterification / esterification of a fatty acid source with an alcohol, to form alkyl fatty acid esters, which comprises the reaction of the said fatty acid source and an alcohol or an alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support and the reaction medium contains an aqueous alkaline buffer solution; and (B) a process for the transesterification / esterification of a fatty acid source selected from triglycerides, diglycerides, monoglycerides and any mixture thereof, said mixture optionally further comprising free fatty acids, with an alcohol, to produce alkyl esters of fatty acids, comprising reacting said source of fatty acid and an alcohol in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support and in which water is added to said fatty acid source or the reaction medium.
    [0002]
    2. Process according to claim 1, characterized by the fact that it is process (A) and that the aqueous buffer solution has a pH of 7 to about 11.
    [0003]
    3. Process, according to claim 1, characterized by the fact that it is process (B) and that the water is in the form of a water solution with a pH of 3 to 11.
    [0004]
    4. Process according to any one of the preceding claims, characterized by the fact that said alcohol is a short-chain alcohol, or that said alcohol donor is a mono-alkyl ester, such as methyl acetate or di -alkyl carbonate, such as di-methyl carbonate, also serving as a source for moderate alkaline reagent in the reaction medium.
    [0005]
    5. Process according to any one of the preceding claims, characterized by the fact that at least one lipase is a lipase derived from any of Rhizomucor miehei, Pseudomonas sp., Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Alcaligenes sp., Acromobacter sp., Burkholderia sp., Thermomyces lanuginosa, Chromobacterium viscosum, Candida antarctica B, Candida rugosa, Candida antarctica A, papaya and pancreatin seeds.
    [0006]
    6. Process according to any one of the preceding claims, characterized by the fact that said immobilized lipase is able to catalyze the esterification of free fatty acids to produce alkyl esters of fatty acid and water as a by-product, and the transesterification of triglycerides and partial glycerides to produce alkyl esters of fatty acid and glycerol as a by-product.
    [0007]
    Process according to claim 1 or 2, in which said is process (A) or the process of any of claims 3 to 6, characterized in that said lipase preparation comprises at least two lipases that each can be separately immobilized on a hydrophobic support or co-immobilized on the same hydrophobic support and that said at least two lipases have identical or different regiospecificity.
    [0008]
    8. Process according to any one of the preceding claims, characterized by the fact that said support is any one of the support based on hydrophobic aliphatic polymer and support based on hydrophobic aromatic polymer.
    [0009]
    Process according to any one of claims 1 to 7, characterized in that said support is porous or non-porous inorganic support, which can be hydrophobic or is coated with hydrophobic organic material.
    [0010]
    10. Process according to any one of the preceding claims, characterized by the fact that said source of fatty acid is any one of vegetable oil, animal fat, algae oil, fish oil, waste oil, brown grease and any mixtures thereof.
    [0011]
    11. Process according to any one of the preceding claims, characterized by the fact that said source of fatty acid comprises free fatty acids, mono-, di- and triglycerides, their mixtures in any proportion, esters of fatty acids and amides, in the absence or presence of other minority fatty acid derivatives, such as phospholipids and sterol esters, said source of fatty acid is unrefined, refined, bleached, deodorized or any of its combinations.
    [0012]
    12. Process according to any one of the preceding claims, characterized by the fact that said alcohol is methanol and the resulting fatty acid esters are methyl fatty acid esters (MEAG - Biodiesel) or that said alcohol is an alcohol medium-chain fatty (C6-C10) or long-chain fatty alcohols (C12-C22).
    [0013]
    13. Process according to any one of the preceding claims, characterized by the fact that said immobilized lipase is used in continuous agitated tank reactors or in filled bed column reactors operating in batch or continuous modes.
    [0014]
    14. System characterized by: (A) a system for the transesterification / esterification of a fatty acid source with alcohol, to form alkyl fatty acid esters comprising a reaction vessel (120) to react a reaction medium including the said fatty acid source and at least one of an alcohol and an alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support and the reaction medium contains an aqueous alkaline buffer solution, or (B) a system for the transesterification / esterification of a fatty acid source selected from triglycerides, diglycerides, monoglycerides and any mixture thereof, said mixture optionally further comprising free fatty acids with an alcohol, for producing alkyl fatty acid esters, it comprises a reaction vessel (120) for reacting a reaction medium including said source of fatty acid and at least one of an alcohol and an alcohol donor in the presence of an immobilized lipase preparation, wherein the immobilized lipase preparation comprises at least one lipase immobilized on a hydrophobic porous support and water is added to said source fatty acids or the reaction medium.
    [0015]
    15. Process according to any one of claims 1 to 13, characterized in that it is conducted in the system as defined in claim 14.
    类似技术:
    公开号 | 公开日 | 专利标题
    BR112012022006B1|2021-02-23|a process for the enzymatic synthesis of alkyl fatty acid esters
    US10689607B2|2020-06-23|Enzymatic transesterification/esterification processes employing lipases immobilized on hydrophobic resins in the presence of water solutions
    US10280389B2|2019-05-07|Enzymatic transesterification/esterification processes employing lipases immobilized on hydrophobic resins in the presence of water solutions
    EP2751275B1|2016-08-03|Enzymatic transesterification with lipases immobilized on hydrophobic resins in water solutions
    IL239404A|2021-08-31|Enzymatic transesterification/esterification processing systems and processes employing lipases immobilized on hydrophobic resins
    BR102013033923A2|2015-05-26|Enzyme transesterification / esterification processing systems and processes employing immobilized lipases in hydrophobic resins
    同族专利:
    公开号 | 公开日
    WO2011107977A1|2011-09-09|
    DK2542685T3|2014-06-23|
    AR080117A1|2012-03-14|
    CN102812128A|2012-12-05|
    ZA201206168B|2014-01-29|
    KR20130004286A|2013-01-09|
    RU2600879C2|2016-10-27|
    JP5554848B2|2014-07-23|
    CA2791836C|2014-10-28|
    KR20170104002A|2017-09-13|
    JP2013520985A|2013-06-10|
    AU2011222439B2|2013-07-18|
    RU2012141298A|2014-04-27|
    AU2011222439A1|2012-09-06|
    MX2012010056A|2012-09-28|
    BR112012022006A2|2019-09-24|
    CA2791836A1|2011-09-09|
    CN102812128B|2017-09-12|
    EP2542685A1|2013-01-09|
    ES2462548T3|2014-05-23|
    EP2542685B1|2014-03-26|
    KR101858915B1|2018-05-16|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5204251A|1987-05-11|1993-04-20|Kanegafuchi Kagaku Kogyo & Kabushiki Kaisha|Process of enzymatic interesterification maintaining a water content of 30-300 ppm using Rhizopus|
    KR0164050B1|1996-03-29|1998-11-16|남창우|Preparation process of ester compounds using enzymes in the absence of solvent|
    JP4012117B2|2002-07-02|2007-11-21|花王株式会社|Method for producing immobilized enzyme|
    DE102004015781A1|2004-03-31|2005-10-20|Cognis Ip Man Gmbh|Process for the enzymatic synthesis of triglycerides of unsaturated fatty acids|
    US7473539B2|2004-09-20|2009-01-06|Sunho Biodiesel Corporation|Methods for producing alkyl esters|
    DE102005002700A1|2005-01-19|2006-07-27|Cognis Deutschland Gmbh & Co. Kg|Compositions usable as biofuel|
    US10119098B2|2005-05-23|2018-11-06|Epax Norway As|Concentration of fatty acid alkyl esters by enzymatic reactions with glycerol|
    CA2621901C|2005-09-12|2016-07-26|Novozymes North America, Inc.|Enzymatic oil interesterification|
    ES2289943B1|2006-07-21|2008-12-16|Universidad De Cordoba|BIODIESEL PRODUCTION PROCEDURE THROUGH THE USE OF PIG PANCREATIC LIPASE AS AN ENZYMATIC BIOCATALIZER.|
    JP5080797B2|2006-12-15|2012-11-21|花王株式会社|Method for producing useful substance using immobilized enzyme|
    IL180598D0|2007-01-08|2007-07-04|Basheer Sobhi|Immobilized interfacial enzymes of improved and stabilized activity|
    IL183084D0|2007-05-09|2007-09-20|Trans Biodisel Ltd|Modified-immobilized enzymes of high tolerance to hydrophilic substrates in organic media|
    US7790429B2|2007-11-28|2010-09-07|Transbiodiesel Ltd.|Robust multi-enzyme preparation for the synthesis of fatty acid alkyl esters|US10000731B2|2010-03-01|2018-06-19|Trans Bio-Diesel Ltd.|Enzymatic transesterification/esterification processes employing lipases immobilized on hydrophobic resins in the presence of water solutions|
    US8308951B1|2010-04-06|2012-11-13|Heliae Development, Llc|Extraction of proteins by a two solvent method|
    MX2012011555A|2010-04-06|2013-01-29|Heliae Dev Llc|Methods of and systems for isolating carotenoids and omega- 3 rich oils from algae.|
    US8475660B2|2010-04-06|2013-07-02|Heliae Development, Llc|Extraction of polar lipids by a two solvent method|
    US8115022B2|2010-04-06|2012-02-14|Heliae Development, Llc|Methods of producing biofuels, chlorophylls and carotenoids|
    US8313648B2|2010-04-06|2012-11-20|Heliae Development, Llc|Methods of and systems for producing biofuels from algal oil|
    US8273248B1|2010-04-06|2012-09-25|Heliae Development, Llc|Extraction of neutral lipids by a two solvent method|
    ES2601802T3|2011-08-31|2017-02-16|Trans Bio-Diesel Ltd.|Enzymatic transesterification with immobilized lipases on hydrophobic resins in aqueous solutions|
    US9200236B2|2011-11-17|2015-12-01|Heliae Development, Llc|Omega 7 rich compositions and methods of isolating omega 7 fatty acids|
    WO2013081446A1|2011-12-02|2013-06-06|Universiti Malaya|A process for producing biodiesel from natural oil and a system thereof|
    US20150353970A1|2012-12-31|2015-12-10|Trans Bio-Diesel Ltd.|Enzymatic transesterification/esterification processing systems and processes employing lipases immobilzed on hydrophobic resins|
    CN103044260A|2013-01-18|2013-04-17|武汉大学|Method for preparation of methyl alkyl carbonate|
    KR101355141B1|2013-03-20|2014-01-29|주식회사 엠알아이|Method for preparing a biodiesel|
    KR101684455B1|2013-07-30|2016-12-08|한국생명공학연구원|A reactor for continuous biodiesel production using immobilized enzyme and method thereof|
    KR101511744B1|2014-04-28|2015-04-22|티케이엘 주식회사|Method for production of cetylated fatty acid complex by enzyme cycling reation|
    CN113293183A|2014-05-28|2021-08-24|诺维信公司|Fatty acid alkyl ester production with base treatment|
    KR101590268B1|2014-09-16|2016-02-01|광운대학교 산학협력단|Method for enzymatic synthesis of glycerol acetate|
    JP2018525020A|2015-08-31|2018-09-06|インターナショナル フローラ テクノロジーズ，リミテッド|Methods and systems for catalytic production of wax ester derivatives|
    US11248245B2|2014-12-19|2022-02-15|International Flora Technologies, Ltd.|Processes and systems for catalytic manufacture of wax ester derivatives|
    CN106906256A|2017-03-20|2017-06-30|威莱日用品有限公司|A kind of synthetic method of fatty acid methyl ester ethoxylate|
    KR102312069B1|2019-12-31|2021-10-13|주식회사 제이엘비|Natural cosmetic containing esters oil|
    KR102332935B1|2020-04-06|2021-11-30|한국조선해양 주식회사|Bio fuel system and marine structure having the same|
    法律状态:
    2019-10-01| B08F| Application fees: application dismissed [chapter 8.6 patent gazette]|
    2020-03-17| B11Z| Dismissal: petition dismissal - article 216, par 2 of industrial property law|Free format text: ARQUIVADA A PETICAO NO 870190110664DE 30/10/2019, UMA VEZ QUE NAO FOI APRESENTADA A PROCURACAO DEVIDA NO PRAZO DE 60 DIAS CONTADOS DA PRATICA DO ATO, CONFORME ART. 216, PARAGRAFO 2O, DA LPI. DESTA DATA CORRE O PRAZO DE 60 DIAS PARA EVENTUAL RECURSO DO INTERESSADO. |
    2020-08-04| B11M| Dismissal: decision cancelled|Free format text: ANULADA A PUBLICACAO CODIGO 11.6.1 NA RPI NO 2567 DE 17/03/2020. |
    2020-08-11| B08G| Application fees: restoration [chapter 8.7 patent gazette]|
    2020-08-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-08-25| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-12-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-02-23| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 23/02/2021, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US30912210P| true| 2010-03-01|2010-03-01|
    US61/309,122|2010-03-01|
    PCT/IL2011/000121|WO2011107977A1|2010-03-01|2011-02-02|A process for the enzymatic synthesis of fatty acid alkyl esters|
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