Batch process for enzymatic modification of lipids

Abstract

The present disclosure relates to a batch process for the enzymatic treatment of a lipid-containing composition, and more particularly, to a batch process for the preparation of a transesterified fat using an enzyme. Advantageously, the processes of the present disclosure may be performed using small amounts of enzymes and have a shorter time range than typical batch enzymatic transesterification processes. The claimed process can be used to replace a batch chemical transesterification process.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 346,489, filed on May 27, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on May 25, 2023 is named '35893_616SEQ' and is 2 kilobytes in size. Background Art

[0005] The present disclosure relates to a batch process for enzymatic treatment of lipid-containing compositions, and more specifically, to a batch process for preparing transesterified fats using enzymes. Advantageously, the process of the present disclosure can be carried out using a small amount of enzymes, and its time range is shorter than the time range of a typical batch enzymatic transesterification process. The claimed process can be used to replace a batch chemical transesterification process.

[0006] Fats are composed of fatty acids attached to a three-carbon glycerol backbone. Fatty acids are composed of a chain of carbon atoms with a terminal hydroxyl group. Hydroxyl groups can be attached to one, two, or three hydroxyl groups on the glycerol backbone to form monoglycerides, diglycerides, or triglycerides or fats. The functional and nutritional quality of fats will depend on a variety of factors, including whether it is a monoglyceride (MAG), diglyceride (DAG) or triglyceride (TAG); the number of carbons in the fatty acid chain; whether the chain is saturated, monounsaturated or polyunsaturated; whether any unsaturated double bonds in the chain are in the form of cis-isomers or trans-isomers; the position of any double bonds along the chain; and the position of different types of fatty acids relative to the three carbons of the glycerol backbone.

[0007] Lipids are a broad class of chemicals characterized by fats, oils, waxes, and phospholipids. This broad classification includes triglycerides, diglycerides, monoglycerides, fatty acids, fatty alcohols, soaps and detergents, terpenes, steroids, and vitamins A, E, D2, and K2. Lipids can be obtained from oilseeds such as soybeans, canola, rapeseed, sunflower, palm, and olives; animal products such as fish, pork, and beef; and synthetic compounds or synthetically derived compositions such as structured lipids for nutritional applications, oleochemicals for industrial and pharmaceutical applications, and biodiesel for energy. Vegetable oils are obtained by pressing or solvent extracting oils from oilseeds. Crude oils contain many minor components. Some of these components are detrimental to the performance or aesthetic properties of the oil; other components, such as sterols and tocopherols, are nutritionally beneficial.

[0008] In recent years, the attention to providing substitutes for high trans fats and shortening products used in traditional food preparation has increased. Traditionally, liquid oils are prepared into solids containing functional fats for various margarines and shortening products by nickel hydrogenation. Such hydrogenation processes lead to the formation of trans fatty acids. It is believed that the fats reduced by trans fatty acids can provide certain health benefits for consumers. Therefore, many large food manufacturers are replacing high trans fats with low trans fats or even zero trans fat compositions. Initially, efforts to provide low trans fat products focused on reducing the hydrogenation level of fat products. Recently, efforts have focused on changing the structure of liquid oils to change melting characteristics and functions without changing the fatty acid composition or producing trans fatty acids. A method of achieving this is a process called transesterification.

[0009] Transesterification is a known reaction of triglyceride structures whereby individual fatty acid structures at various positions of the triglyceride being transesterified are exchanged on the glycerol moiety. This is sometimes referred to or considered as a randomization where the fatty acid moieties of one glycerol component of a triglyceride are interchanged with those of the glycerol component of another triglyceride. This produces triglyceride structures having exchanged fatty acid moieties that vary between glycerol structures.

[0010] Transesterification technology has been developed to produce, for example, triglyceride compositions, which provide certain melting profiles that may be of concern in certain applications. Typically, these are referred to herein as "structured lipids," to distinguish transesterified products from physical blends of the same components that have not yet undergone transesterification. Swem, Bailey "Industrial Oil and Fat Products," the 3rd edition, pp. 941-970 (1964) describes the transesterification of lipids such as fatty acids and glycerol, monohydroxy alcohols and polyhydroxy alcohols, transesterification (acidolysis and alcoholysis) and chemical methods.

[0011] Transesterification can be accomplished chemically or enzymatically. Chemical transesterification (CIE) has been the industry standard for many years and allows the most flexible product mixing and various batch sizes. Chemical transesterification is usually accomplished with chemicals such as sodium methoxide (NaOCH3). CIE reacts rapidly, with the reaction typically taking about 30 minutes, with a batch cycle time of 4 to 8 hours, including loading / drying, chemical dosing and dispersion, reaction endpoint verification, deactivation of the reaction with water or acid solution, and discharge. The reacted oil / fat is moved to a bleaching machine, where most of the undesirable reaction byproducts are removed by silica and / or bleaching clay, and then filtered.

[0012] Although chemical transesterification is rapid, and may be lower than enzymatic transesterification cost in terms of chemicals compared with enzymes, it has several obvious disadvantages. Sodium methylate chemicals are very corrosive and react with water, so they are dangerous and difficult to handle. The CIE process can also produce a large amount of by-products, such as sodium soaps, fatty acid methyl esters (FAME), partial glycerides and process pollutants, and therefore further treatment steps are required to remove undesirable by-products (e.g., water washing to remove a large amount of by-products; using adsorbents to remove undesirable colored compounds produced during the CIE process, etc.). Additionally, if water washing is used for neutralization process, CIE will see an increase in neutral oil loss caused by the emulsion in the separation step. If acid is used, additional losses will be seen in the removal of free fatty acids (FFA) in the physical refining process (deodorization). In general, the basic yield loss calculation of CIE is 10 times of the sodium methylate addition. Therefore, CIE can cause relatively high oil losses, and additional treatment steps need to be used to remove pollutants and by-products.

[0013] Industry is always advancing towards enzymatic transesterification (EIE) technology, because it has selectivity to the hydrolysis of lipid acid (exchange of lipid acid along the glycerol backbone), and can not produce loss or need to bleach the transesterified material again or otherwise handle to remove pollutants and by-products.Enzyme can also undertake that the structure of final transesterified product is carried out a large amount of control.Particularly, use some enzyme to make transesterify at 1-and 3-position along the glycerol backbone, and described position is just the most desirable.Although enzyme catalyst is only used for high value-added product at first, it is now increasingly used in the preparation of commodity fat and fat blend.

[0014] A particularly preferred enzyme catalyst is the lipase from Thermomyces lanuginosus. This enzyme is specific for positions 1 and 3 on the glycerol backbone and has a thermal stability of up to about 75°C. However, this enzyme may be easily inactivated by free radicals such as peroxides, certain polar impurities such as phospholipids and soaps, secondary oxidation products such as ketones and aldehydes, and trace metals. Therefore, the quality of the oil feedstock is important.

[0015] Novozymes Corporation TL IM is a registered trademark for the sale of lipase from Thermomyces lanuginosus in the form of immobilized particles. The product literature accompanying this enzyme product discloses a process for use comprising cooling the lipids to 70°C, pumping the lipids into a single reactor column or tank, and passing the oil through the column or mixing the oil with the enzyme in the tank. The lipids are contacted with the enzyme in the column or tank and transesterification is continuously carried out. The transesterified lipids can then be blended with other lipids, or deodorized, or shipped to the final consumer.

[0016] Although EIE may have certain economic advantages over CIE, some of these economic advantages are associated with the continuous production of a single base stock in a large pipeline flow reactor. However, such designs result in more difficult conversions and mixing, and the packed bed limits flexibility to a certain extent and is best used on large commodity type bases. Additionally, the continuous EIE process requires a large amount of catalyst (enzyme), with about 1 kg of oil / kg of immobilized enzyme used in the process, with a contact time of at least 1 hour. As described in US 8,361,763, in a continuous packed bed reactor system, the total usage rate over the life of the enzyme is 0.4 kg of enzyme per ton of oil processed. Current batch EIE processes also require large amounts of enzyme process use (e.g., about 5% w / w) and long reaction times (typically about 24 hours).

[0017] Therefore, an object of the present disclosure is to provide an enzymatic transesterification process that can replace the traditional CIE process and thereby eliminates the formation of process pollutants after CIE treatment. Another object of the present disclosure is to provide an EIE process that does not require a large amount of process enzyme catalyst and that has a shorter reaction time and greater flexibility than a continuous EIE process. Summary of the invention

[0018] The present disclosure relates to a batch process for enzymatic treatment of lipid-containing compositions, and more specifically, to a batch process for preparing transesterified fats using enzymes. Advantageously, the process of the present disclosure can be carried out using a small amount of enzyme, and its time range is shorter than the time range of a typical batch enzymatic transesterification process. The claimed process can replace a batch chemical transesterification process.

[0019] In one aspect, the present disclosure relates to a process for batch enzymatic treatment of a lipid-containing composition, the process comprising: providing the lipid-containing composition; contacting the lipid-containing composition with an immobilized enzyme material, the immobilized enzyme material comprising a carrier and at least one enzyme immobilized on the carrier, wherein the amount of the immobilized enzyme material is about 1% or less (by weight of the lipid-containing composition); and mixing the lipid-containing composition with the immobilized enzyme material to form an enzymatically treated composition.

[0020] On the other hand, the present disclosure relates to a process for batch enzymatic treatment of multiple lipid-containing compositions, the process comprising: providing a lipid-containing composition; contacting the lipid-containing composition with an immobilized enzyme material, the immobilized enzyme material comprising a support and at least one enzyme immobilized on the support, wherein the amount of the immobilized enzyme material is about 0.72% or less (by weight of the lipid-containing composition); mixing the lipid-containing composition with the immobilized enzyme material to form a first enzymatically treated composition, wherein the mixing occurs for about 10 hours or less; separating the immobilized enzyme material from the first enzymatically treated composition; contacting the immobilized enzyme material with at least one additional lipid-containing composition; and mixing the at least one additional lipid-containing composition with the immobilized enzyme material to form a second enzymatically treated composition. DETAILED DESCRIPTION

[0021] The present disclosure relates to a batch process for enzymatic treatment of lipid-containing compositions, and more specifically, to a batch process for preparing transesterified fats using enzymes. Advantageously, the process of the present disclosure can be carried out using a small amount of enzymes, and its time range is shorter than the time range of a typical batch enzymatic transesterification process. The claimed process can be used to replace a batch chemical transesterification process.

[0022] Therefore, in one aspect, the present disclosure relates to a process for batch enzymatic treatment of a lipid-containing composition. The process comprises providing the lipid-containing composition; contacting the lipid-containing composition with an immobilized enzyme material, the immobilized enzyme material comprising a carrier and at least one enzyme immobilized on the carrier, wherein the amount of the immobilized enzyme material is about 1% or less, or about 0.72% or less (by weight of the lipid-containing composition); and mixing the lipid-containing composition with the immobilized enzyme material to form an enzymatically treated composition. In certain embodiments, the process may further comprise separating the immobilized enzyme material from the enzymatically treated composition.

[0023] The enzymatic treatment described herein may be selected from the group consisting of transesterification, intraesterification, alcoholysis, acidolysis, glycerolysis, transesterification, and combinations thereof. In a specific embodiment, the enzymatic treatment is transesterification.

[0024] Advantageously, the use of the immobilized enzyme materials described herein allows batch enzymatic transesterification to be performed using smaller amounts of enzyme and in a shorter time frame than previously known EIE processes. Additionally, unlike conventional CIE processes, the EIE processes of the present disclosure do not cause the formation of pollutants, and therefore do not require additional treatment of the composition to remove process pollutants and / or byproducts (e.g., water washing), or the use of adsorbents (e.g., silica, activated bleaching clay, activated carbon) for removal of undesirable colored compounds produced in CIE.

[0025] It has been further found that the immobilized enzyme material described herein retains its ability to catalyze EIE reactions even after multiple uses. Without wishing to be bound by any particular theory, it is believed that the combination of enzyme and carrier allows the enzyme to be loaded at a level far higher than other currently available immobilized enzymes. Additionally, it is believed that the orientation of the enzyme on the carrier reduces the enzyme inactivation caused by poisons (i.e., blocking, interfering with the binding site or changing the active site, and / or increasing the thermal stability of the enzyme in the lipid matrix). Therefore, compared with other immobilized enzymes known for EIE processes, the immobilized enzyme material of the present disclosure provides unexpected advantages.

[0026] Therefore, on the other hand, the present disclosure relates to a process for batch enzymatic treatment of multiple lipid-containing compositions. The process comprises providing a lipid-containing composition; contacting the lipid-containing composition with an immobilized enzyme material, the immobilized enzyme material comprising a carrier and at least one enzyme immobilized on the carrier, wherein the amount of the immobilized enzyme material is about 1% or less or about 0.72% or less (by weight of the lipid-containing composition); mixing the lipid-containing composition with the immobilized enzyme material to form a first enzymatically treated composition; separating the immobilized enzyme material from the first enzymatically treated composition; contacting the immobilized enzyme material with at least one additional lipid-containing composition; and mixing at least one additional lipid-containing composition with the immobilized enzyme material to form a second enzymatically treated composition. On the one hand, the lipid-containing composition and / or the at least one additional lipid-containing composition are mixed with the immobilized enzyme material for about 10 hours or less. In certain embodiments, the process may further comprise separating the immobilized enzyme material from the second enzymatically treated composition, and optionally repeating the process one or more additional times by contacting the immobilized enzyme material with one or more additional lipid-containing compositions in the amounts and under the conditions described herein.

[0027] As discussed herein, the process of the present disclosure can be carried out using lower enzyme amounts than are usually required for EIE reactions. In general, the process of the present disclosure comprises contacting a lipid-containing composition with an immobilized enzyme material of the present disclosure in an amount that effectively catalyzes transesterification reactions. In a non-limiting embodiment, the immobilized enzyme material of the present disclosure is used in an amount of about 1 wt.% or less (by weight of the lipid-containing composition). In other embodiments, the amount of the immobilized enzyme material is about 0.72 wt% or less (by weight of the lipid-containing composition). In other embodiments, the amount of immobilized enzyme material is about 0.15wt.% to about 1.0wt.%, or about 0.15wt.% to about 0.72wt.%, or about 0.18wt.% to about 0.72wt.%, or about 0.18wt.% to about 0.5wt.%, or about 0.15wt.% to about 0.48wt.%, or about 0.15wt.% to about 0.4wt.%, or about 0.18wt.% to about 0.36wt.% (by weight of the lipid-containing composition). Unless otherwise stated, the amount of immobilized enzyme material given herein includes the total amount of carrier and enzyme present in the material.

[0028] As discussed herein, in some embodiments, the immobilized enzyme material described herein can be used for more than one enzymatic treatment, including two, three, four or more enzymatic treatments (referred to herein as "used" or "reused" immobilized enzyme material). Thus, in some embodiments, 100% of the immobilized enzyme material used in the enzymatic process can be used immobilized enzyme material. In other embodiments, the immobilized enzyme material used in the process of the present disclosure can be a mixture of used and previously unused (also referred to herein as "fresh") immobilized enzyme material. The total amount of used or used plus fresh immobilized enzyme material used in the process of the present disclosure is about 1 wt.% or less, including about 0.72 wt.% or less, or about 0.15 wt.% to about 1.0 wt.%, or about 0.15 wt.% to about 0.72 wt.%, or about 0.18 wt.% to about 0.72 wt.%, or about 0.18 wt.% to about 0.5 wt.%, or about 0.15 wt.% to about 0.48 wt.%, or about 0.15 wt.% to about 0.4 wt.%, or about 0.18 wt.% to about 0.36 wt.% (by weight of the lipid-containing composition). In embodiments where a mixture of used and fresh immobilized enzyme materials is used, fresh immobilized enzyme material is used in an amount of about 10% to about 75% based on the weight of the used immobilized enzyme material.

[0029] In the embodiment where the fixed enzyme material is used repeatedly, the total amount of the lipid-containing composition processed by the fixed enzyme material is more than the amount of the lipid-containing composition processed with a single batch enzymatic treatment. Therefore, the effective dose of the fixed enzyme material can be less than the dosage of the fixed enzyme material included in the single batch treatment. As used herein, "effective dose" refers to the amount of the fixed enzyme material used by the gross weight of the lipid-containing composition processed in batches. In certain embodiments, the effective dose of the fixed enzyme material used in the process of the present disclosure is about 1wt.% or less, including about 0.72wt% or less, about 0.15wt.% to about 1.0wt.%, about 0.15wt.% to about 0.72wt%, about 0.18wt.% to about 0.72wt.%, about 0.18wt.% to about 0.5wt.%, or about 0.15wt.% to about 0.48wt.%, or about 0.15wt.% to about 0.4wt.%, or about 0.18wt.% to about 0.36wt.%.

[0030] In certain embodiments, the amount of enzyme present in the immobilized enzyme material (referred to herein as "enzyme loading") is from about 3 to about 20% (by weight of the immobilized enzyme material).

[0031] The lipid-containing composition can be contacted with the immobilized enzyme material of the present disclosure at a temperature of about 60° C. to about 100° C., including at a temperature of about 65° C. to about 95° C., about 70° C. to about 95° C., or about 70° C. to about 90° C. In some embodiments, the lipid-containing composition can be contacted with the immobilized enzyme material of the present disclosure at atmospheric pressure. After treatment, the immobilized enzyme material can be separated from the enzymatically treated composition using any suitable technique, including filtration.

[0032] In certain embodiments, the technology provided by this paper relates to and will contain lipid composition and fixed enzyme material and be loaded in the reactor vessel.The reactor vessel can be any industrial reactor type.The reactor vessel can be a container, hydrogenation reactor and / or chemical transesterification reactor designed specifically for batch enzymatic transesterification, such as the stirred tank reactor operated in batch mode.In certain embodiments, this type of container has the member that is used for heating and / or cooling composition during stirring.In certain embodiments, lipid composition and fixed enzyme material can be mixed using any suitable means known in the art, and continue about 10 hours or shorter time, including about 1.5 hours to about 10 hours.

[0033] In certain embodiments, enzymatic treatment is finished in about 10 hours or less time, comprises about 1.5 hours to about 10 hours or about 1.5 hours to about 8 hours or about 10 hours, about 8 hours, about 6 hours, about 4 hours or about 1.5 hours.In a specific embodiment, enzymatic treatment is enzymatic transesterification.When the physical property of oil did not change with the increase of reaction times, realized reaction and finished.In one embodiment, reaction is finished based on the complete randomization of lipid acid on the triglyceride level, and can determine by SFC, dropping point, slip point and the carbon number of the triglyceride level in the measurement oil, as described in the example.

[0034] The lipid-containing composition used in the process of the present disclosure can be a crude oil; refined and bleached; refined, bleached and fully or partially hydrogenated; or fractionated, refined, bleached and deodorized; or any combination thereof. Such compositions can contain fats or oils from plant sources or animal sources. If from a plant source, the oil or fat can be obtained by mechanical pressing or chemical extraction. Oils and fats suitable for use in lipid-containing compositions include, for example and without limitation: canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, meadowfoam seed oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower seed oil, camellia oil, soybean oil, sunflower seed oil, tall oil, tsubaki oil, oil), various "natural" oils with fatty acid compositions altered by genetically modified organisms (GMOs) or traditional "breeding", such as high oleic or low linolenic, low saturated oils (high oleic canola oil, low linolenic soybean oil, or high stearic sunflower oil), vegetable oils, menhaden oil, wax fish oil, cod liver oil, orange crude oil, sardine oil, menhaden oil, lard, tallow, algae oil, fish oil, fats of animal origin, waste cooking oils, brown grease, triglycerides derived from inedible plant sources, partial glycerides and free fatty acids derived from these oils, or any mixture or blend of at least two thereof in any desired ratio.

[0035] Although the method of the present disclosure relates to the batch enzymatic treatment of lipid composition, it should be understood that the immobilized enzyme material used in the process of the present disclosure can also be used in combination with continuous flow application. For example, the immobilized enzyme material can be used to use, for example, a continuous flow reactor to carry out continuous enzymatic treatment of lipid composition. Such a process involves loading the lipid composition and the immobilized enzyme material into a continuous flow reactor, such as a continuous stirred tank reactor (CSTR), a slurry bubbling column or a continuous fixed bed reactor and known variants thereof. In one embodiment, the reactor is a continuous flow fixed bed reactor.

[0036] Immobilized enzyme material

[0037] In order for the immobilized enzyme to be an effective biocatalyst, a certain balance between various properties is important. Specifically, the support should have certain properties that allow the enzyme to be immobilized with high loading and high retention of enzyme activity.

[0038] Suitable pore size is an important characteristic of the carrier. The pore size must be of suitable size so that the enzyme can enter the hole, thereby promoting the fixation of the enzyme in the hole of the whole carrier. Additionally, the carrier must have a sufficiently high surface area so that a sufficiently high enzyme can be loaded onto the surface of the carrier. The pore structure of the carrier should provide a favorable microenvironment without causing complexity due to steric hindrance. The pore structure should produce low solution flow resistance and promote the mass transfer of reactants and products in the whole material.

[0039] Additionally, the chemical properties of the support surface need to facilitate high enzyme loading and additionally provide a chemical environment that enables the enzyme to be retained on the surface of the support and further ensures that the enzyme retains its activity once immobilized on the surface of the support.

[0040] Furthermore, for enzymatic processing of lipid-containing compositions, the support needs to be stable to the reaction conditions and avoid complications caused by swelling due to contact with the lipid or chemical incompatibility with the lipid.

[0041] Thus, in general, in order for an immobilized enzyme to be an effective biocatalyst, a balance of properties such as surface area, pore volume, pore size, chemical functionalization, and chemical stability may be combined to provide an effective biocatalyst suitable for industrial applications.

[0042] In one aspect, the immobilized enzyme material used in the method of the present disclosure comprises:

[0043] a. controlled porosity silica (CPS) as a support having a pore size of about 20 to about 100 nm; wherein the support comprises an amino-functionalized surface; and

[0044] b. One or more catalytically active enzymes immobilized to the amino-functionalized surface via a covalent linker comprising a bond selected from the group consisting of amino, amide, ester, ether, imideamide, imidethioamide, thioether, thioester, and thioamide.

[0045] In a second aspect, the immobilized enzyme material comprises:

[0046] a. controlled porosity silica (CPS) as a support having a pore size of about 20 to about 100 nm; wherein the support comprises an amino-functionalized surface; and

[0047] b. One or more catalytically active enzymes immobilized to the surface via non-covalent interactions.

[0048] In a third aspect, the immobilized enzyme material comprises:

[0049] a. controlled porosity silica (CPS) as a support having a pore size of about 20 to about 100 nm; wherein the support surface comprises at least two different coatings, at least one of the at least two different coatings providing an amino-functionalized surface; and

[0050] b. One or more catalytically active enzymes, optionally immobilized on a surface via a covalent linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester, and thioamide.

[0051] In a fourth aspect, the immobilized enzyme material comprises:

[0052] a. controlled porosity silica (CPS) as a support having a pore size of about 20 to about 100 nm; wherein the support comprises an amino-functionalized surface; and

[0053] b. one or more catalytically active enzymes, optionally immobilized to the amino-functionalized surface via a covalent linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioether, imideamide, imidethioamide, thioester, and thioamide,

[0054] Wherein the enzyme is selected from list (A) disclosed below, preferably from list (B).

[0055] Preferably, the covalent linker in the first, third and fourth aspects (when present) comprises a bond selected from the group consisting of: amino, amide, ester, ether, thioether, thioester and thioamide. More preferably, the covalent linker in the first, third and fourth aspects (when present) comprises a bond selected from the group consisting of: amino, amide, thioether, thioester and thioamide. Even more preferably, the covalent linker in the first, third and fourth aspects (when present) comprises a bond selected from the group having: amino, amide or thioamide. Still more preferably, the covalent linker in the first, third and fourth aspects (when present) comprises a bond selected from the group consisting of: amino, amide and imideamide.

[0056] Preferably, the covalent linker (when present) that immobilizes the enzyme to the surface in the first, third and fourth aspects comprises a bond selected from an amino bond and an amide bond.

[0057] Without being bound by theory, the covalent linker between the amino-functionalized surface and the enzyme contains: (i) a bond formed by the reaction between the amino-functionalized surface and a functional group on the cross-linking reagent used to form the covalent linker, (ii) a bond formed by the reaction between the reactive functional group in the enzyme and the functional group on the cross-linking reagent, and (iii) optionally, any spacer moieties present between the functional groups of the cross-linking reagent, the spacer moieties preferably containing 3-20 atoms comprising any combination of C, N, H and O. The bond referred to as (i) preferably comprises a bond selected from the group consisting of amino, amide and imidoamide. The bond referred to as (ii) may comprise a bond selected from the group consisting of amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester and thioamide.

[0058] Optionally, the immobilized enzyme can be covalently linked intermolecularly by a linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioester, imidoamide, imidothioamide, thioether and thioamide. These bonds are formed in the reaction between the reactive functional groups in the enzyme and the functional groups on the cross-linking agent. The intermolecular linker can optionally comprise a spacer moiety present between the functional groups of the cross-linking agent, the spacer moiety preferably containing 3-20 atoms comprising any combination of C, N, H and O.

[0059] The amino-functionalized surface comprises primary, secondary or tertiary amines.

[0060] Details and preferred features of the immobilized enzyme material are set out below.

[0061] Carrier

[0062] Preferred support materials are inorganic particulate materials having a pore size of about 20 nm to about 100 nm, preferably about 20 to about 60 nm, and more preferably about 30 to about 50 nm.

[0063] WO 2015 / 115993 describes chemically coated controlled porosity glass materials as supports for the preparation of immobilized enzymes with high enzyme loading and high activity. As described in WO 2015 / 115993, the properties of the coated controlled porosity glass materials and the biocatalysts produced therefrom are suitable for the invention described herein, i.e., enzymatic treatment of lipid-containing compositions.

[0064] Another class of supports suitable for the invention described herein is controlled porosity silica (CPS) materials, and specifically amino-functionalized CPS materials. Amino-functionalized CPS supports allow for the immobilization of a variety of different types of enzymes with high enzyme loading, high activity retention, and high stability. The physical properties of amino-functionalized controlled porosity silica materials make them highly suitable for immobilizing enzymes onto support surfaces with high enzyme loading and high enzyme activity retention.

[0065] Controlled porosity silica materials that can be used to prepare amino-functionalized CPS materials are commercially available. For example, controlled porosity silica of the Cariact class can be obtained from Fuji Silysia Chemical Ltd. (https: / / www.fujisilysia.com / products / cariact / ). These inorganic porous silicas can be produced with different surface areas and pore sizes. It has been surprisingly found that these materials are advantageous for fixing enzymes to the surface of a carrier with high enzyme loading. The properties of Fuji Cariact Q-30 and Q-50 make these materials particularly advantageous. Materials Fuji Cariact Q-20C, Q30C and Q40C are also suitable. According to the manufacturer, these particles have the following characteristics:

[0066]

[0067] Another suitable commercially available material is E30 from Ecovyst. The particles have the following properties:

[0068]

[0069] Controlled porosity silica material described herein can be prepared according to the method described in JPH0930809A and JP2020001936. Both methods describe the details of the production method, which provides enough control to modify the properties of silica, thereby producing a controlled porosity silica with necessary properties (such as previously specified surface area, pore volume, pore size and particle size) balance. In addition, these production methods produce controlled porosity silica materials with necessary mechanical strength to achieve the characteristics of the present disclosure. In general, two raw materials, sodium silicate and mineral acid (such as sulfuric acid) are used together in a solution process to produce monomeric silicic acid. Monomeric silicic acid is polymerized to produce primary silica particles known as silica sol. Then, these primary particles are gathered together to form a three-dimensional porous structure. The reaction conditions that enable primary particles to grow and the conditions that dry the product are used to control and modify physical properties, such as surface area, pore size and pore volume.

[0070] The controlled porosity silica materials described herein are specifically designed for use as catalyst supports, in which mechanical properties such as crush strength, wear resistance and abrasion are essential features of the performance of the material as a catalyst support. Chemically and thermally stable controlled porosity silica materials also provide precisely controlled porosity diameters and pore size distributions. Particle sizes and particle size ranges can be customized and optimized for different industrial reactor types. For example, it can be deployed in batches or continuously stirred tank reactors (CSTRs) as slurry catalysts or slurry bubble columns or fixed bed reactors or known variants thereof.

[0071] The appropriate pore size provides a favorable microenvironment without the complexity caused by steric hindrance. The pore structure produces low solution flow resistance and promotes mass transfer of reactants and products throughout the material. The rigid non-cage structure of CPS provides a rugged, incompressible medium that is suitable for high-throughput reactor design and linear scale-up at high flow rates. The material exhibits limited swelling in solvents and is chemically and dimensionally stable in most organic media and aqueous environments with a pH below 10.

[0072] Generally, the pore size of the controlled porosity silica support suitable for the present invention is from about 20 nm to about 100 nm, preferably from about 20 to about 60 nm, and more preferably from about 30 to about 50 nm. Preferably, the surface area is about 50 m 2 / g to about 200m 2 / g (preferably greater than about 60m 2 / g, such as greater than about 70m 2 / g). And preferably, the pore volume is from about 0.5 mL / g to about 2.0 mL / g (more preferably at least about 0.6 mL / g, even more preferably at least about 0.7 mL / g, and most preferably from about 0.8 mL / g to about 1.5 mL / g). These properties allow the enzyme to be immobilized on the surface of the support at high loadings. Preferably, the support has a pore size of about 20 to about 60 nm and a surface area of ​​about 50 m 2 / g to about 200m 2 Even more preferably, the carrier has a pore size of about 20 to about 60 nm and a surface area of ​​about 50 m 2 / g to about 200m 2 / g, and a pore volume of about 0.5 mL / g to about 2.0 mL / g. Most preferably, the support has a pore size of 20 to 60 nm and a surface area of ​​50 m 2 / g to 200m 2 / g, and the pore volume is 0.5mL / g to 2.0mL / g. The ranges disclosed herein include the endpoints.

[0073] CPS supports exhibit an enzyme loading capacity that is inversely related to their pore size. Thus, CPS supports with large pore sizes cannot load as much enzyme as CPS supports with smaller pore sizes, primarily due to the inverse relationship between pore size and surface area (i.e., lower available surface area for enzyme immobilization).

[0074] The preferred particle size of the carrier depends on the type of reactor deployed for use with the fixed enzyme material. For example, a smaller particle size (e.g., 75-150 μm) is well suited for deployment in batches or continuous stirred tank reactors, in which the fixed enzyme material is operated as a catalyst slurry in the reactor. For deployment of fixed bed reactors, the preferred particle size depends on the chemical reaction and conditions in the reactor. For liquid phase fixed bed reactions, smaller particles (e.g., 75-150 μm) are preferred, and for some other liquid phase reactions and reactions in which gas and liquid are deployed together in fixed bed reactors, a larger particle size can be deployed, such as a particle size greater than 200 μm, and preferably greater than 500 μm.

[0075] The amino-functionalized CPS supports are chemically stable, which means that they can be used in a wide range of reaction conditions, including reactions involving substrates such as lipids.

[0076] It should be understood that CPS in this context is different from siliceous mesoporous foams (MCFs) known in the literature. MCFs have an ordered 3D cage-like structure with spherical cavities of 20-40 nm in diameter interconnected by pores of about 10 nm in size, which are smaller than desired for enzyme immobilization. In contrast, CPSs are not cage-like structures. In the context of MCF materials, the literature generally uses the term pore size to define the diameter of the cage and the pore window to define the cage entrance diameter (or pore size), where the window size is generally smaller than the pore size. Due to the cage-like structure and the smaller window size, the surface area (m 2 / g) is usually significantly higher than the surface area of ​​CPS (over 200m 2 MCF is currently not commercially available on an industrial scale and is too expensive for industrial applications, so it is not an operationally viable alternative to CPS.

[0077] Controlled porosity silica is also different from hollow microsphere silica described in the literature. WO 2013 / 078551 describes hollow microsphere silica as microcapsules comprising a silica shell having a thickness of about 50 nm to about 500 μm surrounding a hollow capsule having a diameter of about 0.1 μm to about 1500 μm and a density of about 0.001 g / cm 3 To about 1.0g / cm 3. Figures 2-5 of WO 2013 / 078551 clearly show the hollow features of these materials. These materials are core / shell / functional surface type reservoirs or microcapsules, comprising a core (gaseous or hollow), surrounded by a shell (usually solid), the shell being mainly composed of one or more silica-based materials, and being terminated by a functional surface having affinity or adhesion to a matrix of a plastic or composite material or rubber or textile. The microcapsules are designed to be introduced into the products during the processing stage of plastic, composite material, rubber and textile products. Gaseous or hollow microcapsules are dispersed throughout or partially in plastic, composite material, rubber and textile products as density-reducing additives to reduce the density of the final product. While the hollow sphere microcapsules of WO2013 / 078551 are designed as density-reducing additives, the density-reducing additives can provide material advantages for the formulated products, the controlled porosity silica material of the present invention is specifically designed for use as a catalyst carrier, in which mechanical properties such as crushing strength, wear resistance and abrasion are essential features of the performance of the material as a catalyst carrier. Such low density, hollow sphere supports, which inherently have low crush strength, would not provide such properties. The controlled porosity silica of the present invention is not hollow and comprises a three-dimensional silica structure comprising precisely controlled pore sizes and pore size distributions throughout the solid, and is well suited for the synthesis of efficient and robust biocatalysts.

[0078] Amino-functionalized surfaces

[0079] The amino-functionalized surface may comprise a structure of formula (I), (II) or (III):

[0080]

[0081] in

[0082] R 1 It is C 1-6 alkanediyl; and wherein R 1 Provides covalent attachment to the silica surface (vertical line on the left);

[0083] X is a bond (i.e., X is absent) or a phenyl ring;

[0084] R 2 It is C 1-6 Alkanediyl;

[0085] R 3 It is W;

[0086] R 4 is W or selected from the group consisting of: C 1-10 Alkyl, C 5-7 Cycloalkyl, phenyl, phenyl-C 1-6-alkyl, amino-C 2-8 Alkyl, N-(phenyl)amino-C 2-8 Alkyl and N-(phenyl-C 1-6 -alkyl)amino-C 2-8 alkyl and aminocarbonyl; and wherein the phenyl group is in each case optionally substituted by one or more substituents C 1-4 Alkyl substitution;

[0087] or R 3 and R 4 is combined with the nitrogen atom to which it is attached to form a 5- to 7-membered heterocyclic ring, which may contain one or more unsaturated bonds, and which may contain one or more additional heteroatoms, to which is optionally attached a covalent linker comprising a bond selected from the group consisting of amino, amide and imidoamide, wherein the catalytically active enzyme is also attached to the linker; or R 3 and R 4 Together they form = CR 5 R 6 , where R 5 and R 6 Each is independently C 1-4 alkyl,

[0088] Each R 7 Independently C 1-6 alkanediyl; and wherein R 7 Provides covalent attachment to the silica surface (vertical line on the left);

[0089] R 8 It is C 1-3 Alkanediyl;

[0090] Each R 9 is independently W or selected from C 1-4 Alkyl and hydroxy-C 1-4 A group consisting of alkyl groups;

[0091] Each R 10 Independently C 1-6 alkanediyl; and wherein R 10 Provides covalent attachment to the silica surface (vertical line on the left);

[0092] R 11 is W or selected from the group consisting of: C 1-10 Alkyl, C 5-7 Cycloalkyl, phenyl, phenyl-C 1-6 -alkyl, amino-C 2-8 Alkyl, N-(phenyl)amino-C 2-8 Alkyl and N-(phenyl-C 1-6 -alkyl)amino-C 2-8alkyl and aminocarbonyl; and wherein the phenyl group is in each case optionally substituted by one or more substituents C 1-4 Alkyl substituted; and

[0093] Each W is independently hydrogen or a covalent linker comprising a bond selected from amino, amide and imidoamide, wherein the catalytically active enzyme is also attached to the linker.

[0094] In some embodiments, the amino-functionalized surface comprises a structure of formula (I), as defined above, in combination with a structure of formula (IV):

[0095]

[0096] Where R 12 Selected from C 1-12 Alkyl and amino-C 1-12 Preferably, the amino functional groups in the surface are not part of an aromatic heterocycle, more preferably not any heterocycle. Preferably, the surface does not contain any functional groups other than amino groups that can chelate metal ions. More preferably, the surface comprises amino functionalized aliphatic moieties.

[0097] In some cases, it is beneficial to have at least two different coatings on the surface of the support (at least one of the at least two different coatings provides amino functionalization). For certain enzymes, the presence of two different coatings can provide a better balance of properties suitable for the certain enzymes, resulting in a more effective biocatalyst. For example, a certain enzyme may prefer a coating that is more hydrophobic in nature and may also benefit from the presence of amino groups and additionally metal ions. The presence of at least two coatings can accommodate such situations, one coating providing amino functionalization and the other providing a hydrophobic coating. The hydrophobic coating may contain alkyl moieties and / or aromatic moieties. Preferably, the hydrophobic coating contains C 1-12 Alkyl groups, preferably containing C 1-6 Alkyl and / or phenyl.

[0098] For example, one coating may contain aliphatic chains or phenyl groups, thereby providing a hydrophobic environment, and the second may contain amino functional groups, which can further accommodate metal ions. The relative proportions of the two coatings on the surface can be controlled and optimized to maximize the efficiency of the biocatalyst. On a weight basis, the relative amounts of the two coatings may range from 9:1 to 1:9, preferably 5:1 to 1:5, more preferably 2:1 to 1:2, and most preferably 1:1.

[0099] Additionally, the presence of two coatings can be beneficial in situations where covalent bonding between the enzyme and the surface of the CPS material is preferred. The presence of two coatings can tailor a surface with two coatings that can a) provide chemical groups preferred by the enzyme, such as hydrophobic groups such as aliphatic chains or phenyl groups, and b) provide amino functional groups to enable covalent bonding between the enzyme and the CPS surface.

[0100] Preferably, the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide, and imidoamide, wherein the catalytically active enzyme is also attached to the linker:

[0101]

[0102] In some embodiments, the amino-functionalized surface comprises structure (I'):

[0103]

[0104] in

[0105] R 4 is W or is selected from the group consisting of: phenyl, phenyl-C 1-6 -alkyl, amino-C 2-8 Alkyl, N-(phenyl)amino-C 2-8 Alkyl and N-(phenyl-C 1-6 -alkyl)amino-C 2-8 alkyl, and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and

[0106] W is hydrogen or a covalent linker comprising a bond selected from the group consisting of amino, amide, and imideamide, wherein the catalytically active enzyme is also attached to the linker;

[0107] or wherein the amino-functionalized surface comprises structure (I″):

[0108]

[0109] in

[0110] R 4 is W or selected from the group consisting of: amino-C 2-8 Alkyl, N-(phenyl)amino-C 2-8 Alkyl and N-(phenyl-C 1-6 -alkyl)amino-C 2-8 alkyl, and wherein the phenyl group is optionally substituted by one or more substituents C 1-4 Alkyl substituted; and

[0111] W is hydrogen or a covalent linker comprising a bond selected from amino, amide and imidoamide, wherein the catalytically active enzyme is also attached to the linker.

[0112] In certain preferred embodiments, the amino-functionalized surface comprises structure I′, wherein R 4 is W or is selected from the group consisting of phenyl, benzyl, amino-C 2-6 Alkyl, N-(phenyl)amino-C 2-6 Alkyl and N-(benzyl)amino-C 2-6 Alkyl; or amino functionalized surface comprises structure I″, wherein R 4 It's W.

[0113] In some embodiments, the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide, and imidoamide, wherein the catalytically active enzyme is also attached to the linker:

[0114]

[0115] Most preferably, the amino-functionalized surface comprises any of the following structures, wherein each W is hydrogen or a covalent linker comprising a bond selected from amino, amide, and imidoamide, wherein the catalytically active enzyme is also attached to the linker:

[0116] (1) Propylamine

[0117]

[0118] (2) Propyl-NH-N-aminoethyl

[0119]

[0120] (3) Propyl-NH-N-aminohexyl (LCAA)

[0121] or

[0122] (4) Phenethyl-methyl-NH-N-aminoethyl

[0123]

[0124] For the avoidance of any doubt, it should be understood that the names of the above structures (1)-(4) are derived from the case where W=H, but however, the structures also encompass the case where W is a covalent linker comprising a bond selected from amino, amide and imidoamide, wherein the catalytically active enzyme is also attached to the linker.

[0125] As used herein, the term “C 1-6"Alkanediyl" refers to a straight-chain or branched divalent group having 1 to 6 carbon atoms. 1—6 Examples of the alkanediyl group include a methanediyl group, an ethanediyl group, a 1,3-propanediyl group, and a 2,2-dimethyl-1,4-butanediyl group.

[0126] As used herein, the term “C 1-10 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 10 carbon atoms, and the term "C 1-4 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms. 1-4 Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0127] As used herein, the term “C 5-7 "Cycloalkyl" refers to a monocyclic saturated hydrocarbon ring having 5 to 7 carbon atoms. 5-7 Examples of cycloalkyl groups include cyclopentyl, cyclohexyl and cycloheptyl.

[0128] As used herein, the term "hydroxy-C 1-4 "Alkyl" refers to a C 1-4 alkyl.

[0129] As used herein, the term "phenyl-C 1-6 "Alkyl" refers to a C 1-6 alkyl.

[0130] As used herein, the term "amino-C 2-8 "Alkyl" refers to a C 2-8 alkyl.

[0131] The term "amino" refers to primary, secondary, and tertiary amines.

[0132] The term "aminocarbonyl" refers to the group -C(=O)NH2.

[0133] As used herein, the term "N-(phenyl)amino-C 2-8 Alkyl" and "N-(phenyl-C 1-6 -alkyl)amino-C 2-8 "Alkyl" refers to an amino-C1-6 alkyl group in which one of the hydrogen atoms on the amino group is replaced by a phenyl group or a phenyl-C1-6 alkyl group. 2-8 alkyl.

[0134] As used herein, the term surface in the context of the immobilized enzyme materials described herein refers to the entire surface of the CPS support and includes both the external surface and the pore surface. Due to the nature of the CPS, most of the surface area is present in the pores.

[0135] Immobilized enzyme

[0136] Any enzyme of interest may be used in the presently disclosed process. For example, the enzyme used in the process of the invention may be a lipase; an esterase; an acylase; a transferase; an oxidoreductase; a hydrolase; a ligase; an isomerase; a lyase; those enzymes that promote acidolysis, transesterification, ester synthesis, or transesterification; enzymes having phospholipase or protease activity (including thermostable and thermotolerant hydrolase activity); and polynucleotides. Suitable enzymes include, but are not limited to, those derived from Achromobacter, Alcaligenes, Aspergillus, Bacillus, Burkholderia, Candida, Chromobacterium, Corynebacterium, Fusarium, Geotrichum, Humicola, Humicola, Mucor, Penicillium, Pseudomonas, Rhizomucor, Rhizopus, Staphylococcus, Thermomyces, or Torulopsis. Suitable derivatizing enzymes include, but are not limited to, Aspergillus niger Mucor mihei, Mucor javanicus Pseudomonas Fuorescens, Rhizopus delemar, Burkholderia cepacia, Candida cylindracea, Candida Antarctica, Candida rugosa, Fusarium solani, Penicillium cyclopium, Rhizomucor miehei, and Thermomyces lanuginosus.

[0137] In one embodiment, the immobilized enzyme can be selected from the enzymes in the list (A) consisting of items 1-62 in Table A below and their enzymatically active sequence variants, preferably variants with similar enzymatic activity, and most preferably variants with substantially the same enzymatic activity.

[0138] As is well known, the amino acid sequence of enzyme can have considerable variation without significantly affecting enzymatic activity. For example, artificial sequences such as His tags can be added to realize metal chelation. In other cases, residues that are not critical to enzymatic activity can be modified to improve properties (such as stability or production efficiency), or to circumvent patent protection by only providing alternative sequences. In some cases, the enzyme sequence can be truncated to produce a more compact enzyme by eliminating the part that is not critical to enzymatic activity in the enzyme. Therefore, the enzymatic activity sequence variants of the enzyme identified in Table A can also be used, and therefore covered by List (A).

[0139] Table A: Preferred immobilized enzymes

[0140]

[0141]

[0142]

[0143] Reference 1: Global Substance Registration System version 3.0.3, UNII record A370TYK9KO

[0144] Reference 2: Cuetos et al. (2016) Angew Chem Int Ed 2016, 55: 3144-3147

[0145] The immobilized enzyme is preferably selected from the list (B) consisting of: Thermomyces lanuginosus lipase (TLL, No. 1 in Table A), Candida antarctica lipase (CalB, No. 2 in Table A), Bifidobacterium adolescentis sucrose phosphorylase (SucP, No. 3 in Table A), Thermoanaerobacter buchneri secondary alcohol dehydrogenase (TbSADH, No. 4 in Table A), Arthrobacter amine aminotransferase (ATA, No. 60 in Table A), Mesentericum glycosyltransferase (GT, No. 18 in Table A), amylotransferase from Aspergillus niger (No. 61 in Table A) and aminotransferase (Arthrobacter) (No. 62 in Table A), as well as enzymatically active sequence variants thereof, preferably variants having similar enzymatic activity, and most preferably variants having substantially the same enzymatic activity.

[0146] A particularly preferred enzyme catalyst is a lipase from Thermomyces lanuginosus. The Thermomyces lanuginosus lipase having SEQ ID NO: 1 can be used in liquid form as 100 L (Novozymes).

[0147] The "EC number" column in Table A indicates the enzyme activity that is relevant in the context of Lists (A) and (B) and refers to the Enzyme Commission's numerical classification scheme, which classifies enzymes according to the reaction they catalyze.

[0148] The Uniprot id column in Table (A) refers to entries in the public Uniprot database at www.uniprot.org as of December 1, 2021. Preferably, each enzyme selected in List (A) or List (B) has at least 70% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, still more preferably at least 95% sequence identity, and most preferably complete sequence identity with the sequence of the identified database entry indicated by "Uniprot id" in Table A.

[0149] In some embodiments, the enzyme comprises a metal affinity tag, such as a poly-histidine tag, an HQ tag, a MAT tag, or any other suitable tag known in the art, to facilitate immobilization by metal chelation.

[0150] Enzyme immobilization

[0151] In preferred embodiments of the third and fourth aspects, the enzyme is immobilized to the surface via a covalent linker comprising a bond selected from the group consisting of amino, amide and imide amide, although non-covalent immobilization is also an option.

[0152] In preferred embodiments of the first, third and fourth aspects, the enzyme is fixed to the surface via a covalent linker comprising a bond selected from amino, amide and imide amide, and prior to forming the bond, the enzyme has been fixed to the surface via non-covalent interactions, preferably via interactions mediated by chelated metal ions. Without being bound by theory, fixation after non-covalent binding may allow for more uniform crosslinking between the CPS support surface and the enzyme, thereby improving activity retention. In other embodiments, the initial fixation is via a covalent linker, optionally followed by intermolecular crosslinking.

[0153] In preferred embodiments of the second, third and fourth aspects, the enzyme is non-covalently immobilized to the surface via interactions mediated by chelated metal ions.

[0154] The chelated metal ions referred to above can be selected from Ni 2+ , Cu 2+ Mg 2+ , Fe 3+ or Zn 2+ Preferably, the metal ion is Zn2+ Ions. This type of Zn 2+ Ion-mediated interactions provide superior results for some enzymes such as CalB and TbSADH.

[0155] In certain embodiments of the first, second, third or fourth aspect, the immobilized enzyme is covalently linked between molecules via a linker comprising a bond selected from the group consisting of amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester and thioamide.

[0156] Preferably, the covalent linker between the surface and the enzyme (if present) does not comprise an imine bond that can be easily cleaved by hydrolysis. Preferably, the intermolecular covalent linker (if present) does not comprise an imine bond. Preferably, the covalent linker (if present) comprises a bridge containing 3 to 20 atoms comprising any combination of C, H, N and O.

[0157] In some embodiments, the enzyme comprises a metal affinity tag, such as a polyhistidine tag (a His tag of 2-8 consecutive histidines, preferably 6 consecutive histidines), an HQ tag, a MAT tag, or any other suitable tag known in the art, to facilitate immobilization by metal chelation. It should be noted that many industrially relevant enzymes can be immobilized by metal chelation even without affinity tags due to the inherent metal chelation properties of enzymes. However, compared with physical adsorption, the advantage of immobilization by metal chelation using affinity tags is selective enzyme binding, and in addition, the spatial orientation of the immobilized enzyme can be more uniform compared to the case where immobilization occurs, for example, by non-specific physical adsorption. Affinity tags can also improve post-immobilization activity by driving fixed positions, thereby allowing better and / or more consistent access to active sites. On the other hand, initial immobilization by covalent linkers can also be applied because it can afford a simplified preparation process because the non-covalent fixation step can be omitted.

[0158] Covalent cross-linking (between different enzyme molecules and / or between enzyme molecules and amino groups of amino-functionalized surfaces) has certain advantages. Cross-linking allows for better retention of enzyme activity under industrial flow conditions and the potential for recycling and reuse of catalysts in batch operations.

[0159] Suitable cross-linking reagents for forming covalent cross-links are disclosed below.

[0160] Cross-linking reagents

[0161] Various covalent cross-linking strategies applied to amino functional groups and enzymes are known in the field of protein chemistry. See, for example, Crosslinking Technical Handbook, published by Thermo Scientific, which is incorporated herein by reference, https: / / tools.thermofisher.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf.

[0162] An effective cross-linking reagent for connecting the enzyme to the surface of the amino-functionalized carrier will require at least two (preferably no more than two) reactive groups separated by a joint. An effective cross-linking reagent needs to contain at least a first reactive group, which can react with the amino-functional group attached to the CPS surface to form a stable covalent bond. Additionally, the cross-linking reagent needs to contain at least a second reactive group, which can react with the functional group present on the enzyme structure to form a stable covalent bond, thereby establishing a covalent connection between the enzyme and the carrier surface. Alternatively, the reagent can contain at least two reactive groups, each of which can react with the functional group present on the enzyme structure to form a stable covalent bond, thereby establishing an intermolecular covalent connection between the enzyme molecules.

[0163] Functional groups suitable for cross-linking on enzyme include amine, carboxylate group, thiol (mercapto) and hydroxyl. In the case of selecting appropriate cross-linking reagent, these functional groups can easily form covalent bonds, such as amino, amide, ester, ether, thioether, imidoamide, imidothioamide, thioester and thioamide. As a specific example, in the case of epoxide cross-linking reagent, the functional group on enzyme can react with epoxide group to produce covalent bond, such as amino bond, ester bond, ether bond and thioether bond. For example, in the case of diepoxide cross-linking reagent, one epoxide group in epoxide group can react with amino functional group on the surface of amino functionalized controlled porosity silica carrier to form amino covalent bond. The second epoxide group can react with the functional group located on the amino acid residue positioned on enzyme. For example, the amino functional group from lysine amino acid residue can react with epoxide group to form amino covalent bond. Alternatively, the thiol functional group from cysteine ​​amino acid residue can react with epoxide group to form thioether covalent bond. In yet further embodiments, a carboxylate functional group from a glutamic acid amino acid residue can react with an epoxide group to form an ester covalent bond, or a hydroxyl group from a serine amino acid residue can form an ether covalent bond.

[0164] If desired, cross-linking reagents can similarly be used to form inter-molecular linkers between immobilized enzymes.

[0165] The cross-linking reagent can be homofunctional or heterofunctional, which means that the first reactive group and the second reactive group can be the same or different. Preferably, the first reactive group and the second reactive group are separated by a linker containing 3 to 20 atoms comprising any combination of C, H, N and O.

[0166] An effective cross-linking agent should establish a covalent bond between the enzyme and the support surface and retain enzyme activity after cross-linking. Enzyme activity retention after cross-linking is an important desired feature of cross-linking reagents, which can be adjusted by selecting appropriate reactive groups, linker groups, and linker lengths.

[0167] The preferred reactive group selected is to react with amine, hydroxyl, carboxylate or thiol, to select with the ability of setting up stable covalent bond by amino, amides, ether, ester, imide amide, imide sulfamide, thioether, thioester or thioamide bond.Can contain at least two reactive groups with the cross-linked cross-linking reagent of the functional group of enzyme, and described at least two reactive groups are independently selected from epoxide, ester, acid anhydride, N-hydroxysuccinimide ester, imide ester, carbonate, acyl isourea, carbodiimide, maleimide, halogenated acetyl, thiosulfonate, isocyanate and vinyl sulfone.Reactive groups such as carbodiimide can react with for example carboxylate group etc., and then self stand by second reactive group displacement to form covalent bonds such as amido bond to help form covalent bond.

[0168] The following formula defines a preferred cross-linking reagent:

[0169]

[0170] wherein Y and Z are independently reactive groups selected from the group consisting of epoxides, esters, anhydrides, imide esters, N-hydroxysuccinimide esters, carbonates, acylisoureas, carbodiimides, maleimides, haloacetyl groups, thiosulfonates, vinyl sulfones and isocyanates, and preferably, Y and Z are each independently epoxides, N-hydroxysuccinimide esters or imide esters;

[0171] L represents a linker between reactive groups containing between 3 and 20 atoms including any combination of C, H, N and O.

[0172] More preferred cross-linking reagents are homofunctional, wherein Y = z, and contain a reactive group selected from the group consisting of epoxides, esters, anhydrides, N-hydroxysuccinimide esters, imido esters, carbonates, acylisoureas, maleimides, haloacetyl groups, and vinyl sulfones, most preferably epoxides, N-hydroxysuccinimide esters, or imido esters.

[0173] Even more preferably, the cross-linking agent is a diepoxide agent, still more preferably a diepoxide defined by the formula:

[0174]

[0175] wherein L represents a linker between epoxide groups containing 3 to 20 atoms including any combination of C, H, N and O.

[0176] Most preferably, the diepoxide reagent is glycerol diglycidyl ether (GDE, which may be 1,3- or 1,2-substituted glycerol diglycidyl ether, or a mixture of these two isomers), neopentyl glycol diglycidyl ether (NPE), poly(tetraethylene oxide) diglycidyl ether (PDE), 1,6-hexanediol diglycidyl ether (HDDE) or glycerol triglycidyl ether (GTGE).

[0177] Preferred alternative cross-linking agents include difunctional sulfonated N-hydroxysuccinimide esters and difunctional imide esters, such as bis(sulfosuccinimidyl) suberate (BS3) or dimethyl suberate (DMS).

[0178] A known alternative cross-linking agent is glutaraldehyde, but it results in the formation of imine bonds, so it is not covered by the present invention. Glutaraldehyde is a particularly reactive cross-linking agent that results in a large degree of enzyme inactivation. Imine bond formation is also reversible, so the connection is not very stable.

[0179] Preferred properties of enzyme immobilization materials

[0180] In a preferred embodiment of the first aspect, the support material has a pore size of about 20 to about 60 nm and a surface area of ​​about 50 m 2 / g to about 200m 2 / g, and the pore volume is from about 0.5 mL / g to about 1.5 mL / g, and the enzyme is selected from list (A), or even more preferably from list (B).

[0181] In a preferred embodiment of the second aspect, the support material has a pore size of about 20 to about 60 nm and a surface area of ​​about 50 m 2 / g to about 200m 2 / g, and the pore volume is from about 0.5 mL / g to about 1.5 mL / g, and the enzyme is selected from list (A), or even more preferably from list (B).

[0182] In a preferred embodiment of the third aspect, the support material has a pore size of about 20 to about 60 nm and a surface area of ​​about 50 m 2 / g to about 200m 2 / g, and the pore volume is from about 0.5 mL / g to about 1.5 mL / g, and the enzyme is selected from list (A), or even more preferably from list (B).

[0183] In some embodiments, after fixation, the immobilized enzyme retains at least 20%, preferably at least 30% of its activity compared to the state before fixation; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, yet more preferably at least 70%, further more preferably at least 80%, and most preferably at least 90%. In rare cases, fixation can even enhance the activity so that the apparent retention rate exceeds 100%. In some embodiments, after fixation, the immobilized enzyme retains 20%-100%, preferably 30%-100% of its activity compared to the state before fixation; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, yet more preferably 70%-100%, further more preferably 80%-100%, and most preferably 90%-100%.

[0184] In some embodiments, the immobilized enzyme retains at least 20%, preferably at least 30% of its activity after 20 hours under continuous flow conditions compared to the state before immobilization; more preferably at least 40%, even more preferably at least 50%, still more preferably at least 60%, yet more preferably at least 70%, further more preferably at least 80%, and most preferably at least 90%. In some embodiments, the immobilized enzyme retains 20%-100%, preferably 30%-100% of its activity after 20 hours under continuous flow conditions compared to the state before immobilization; more preferably 40%-100%, even more preferably 50%-100%, still more preferably 60%-100%, yet more preferably 70%-100%, further more preferably 80%-100%, and most preferably 90%-100%.

[0185] sequence

[0186] SEQ ID NO: 1 = Thermomyces lanuginosus lipase

[0187]

[0188] Examples

[0189] The process of the present disclosure is further illustrated by the following examples. These examples should not be viewed in a limiting sense.

[0190] Materials and methods

[0191] Analytical methods

[0192] Solid Fat Content (SFC) - Official Method of the American Oil Chemist's Society, Cd 16b-93, Revised 2017.

[0193] Iodine Value - Official Method of the American Oil Chemists Association, Cd 1d-92, reapproved 2017.

[0194] Dropping Point - Official Method of the American Oil Chemists Association, CC 18-80, reapproved 2017.

[0195] Triglycerides in vegetable oils by HPLC (carbon number) - Official Method of the American Oil Chemists Association, CE 5-86, revised 2017.

[0196] Experimental equipment

[0197] Unjacketed 1.2L glass reaction vessel; 150mm Schott O-ring; 12mm drain valve; 12mm PTFE stem and control knob on bottom for draining and sample removal.

[0198] 4-hole cap with clamp and O-ring for Duran reaction vessel.

[0199] Jacketed electric heated fireplace.

[0200] Temperature controller, J-Kem Scientific - Gemini.

[0201] Overhead glass stirrer with Teflon blade and stirrer bearing (Teflon).

[0202] Temperature probe - K type.

[0203] Vacuum pump.

[0204] Buchner Filter, 70 mm.

[0205] Wattman filter paper No. 1, 70 mm

[0206] 2L vacuum flask

[0207] Hot plate with magnetic stirrer

[0208] Magnetic stirring bar, 20 mm

[0209] Enzymes

[0210] TL-IM - a lipase in the form of immobilized particles from Thermomyces lanuginosus having SEQ ID NO: 1. The Thermomyces lanuginosus lipase is a 1,3 specific lipase immobilized on a silica gel support and formed into immobilized particles. TL-IM is commercially available from Novozymes. The label of TL-IM indicates that the maximum operating temperature is 70°C.

[0211] Test enzyme TL 100L, a Thermomyces lanuginosus lipase having SEQ ID NO: 1, was purchased from Novozymes and immobilized on a carrier as described in Example 2.

[0212] Test enzyme TL 100L, a Thermomyces lanuginosus lipase having SEQ ID NO: 1, was purchased from Novozymes and immobilized on a carrier as described in Example 3.

[0213] Oil

[0214] The oils used in the examples were 85:15 or 65:35 fractionated palm stearin and palm kernel oil blends. Table 1 below lists the properties of the 85:15 fractionated palm stearin / palm kernel oil blend before enzymatic treatment (unreacted), and the target profile after enzymatic treatment (reacted). Tables 2 and 2a list the profiles of fractionated palm stearin, palm kernel oil, and 65:35 fractionated palm stearin / palm kernel oil blends before enzymatic treatment.

[0215] Table 1: Profile of the 85:15 fractionated palm stearin / palm kernel oil blend used in Trials 1 to 11.

[0216]

[0217] Table 2: Overview of fractionated palm stearin, palm kernel oil and 65:35 fractionated palm stearin / palm kernel oil blend used for runs 12 to 25.

[0218]

[0219] Table 2a: Overview of fractionated palm stearin oil, palm kernel oil and 65:35 fractionated palm oil / palm kernel oil blend used for runs 26 to 36.

[0220]

[0221]

[0222] Example 1: Batch enzymatic transesterification

[0223] In this example, use TL-IM or immobilized Thermomyces lanuginosus lipase prepared as described in Example 2 ("Test Enzyme I") and Example 3 ("Test Enzyme II"), subjected 85:15 or 65:35 fractionated palm oil and palm kernel oil blends to batch enzymatic interesterification under different reaction conditions.

[0224] Test 1: 0.12 wt% at 70°C TL-IM; 85:15 blend

[0225] A 1700 g sample of 85:15 fractionated palm oil / palm kernel oil blend was added to a 2 liter reaction vessel. The temperature was set to 70°C and heated to reaction temperature with stirring. A 0.12 wt% dose (by weight of oil) was added to the mixture. TL-IM is added to the reaction vessel through the open port. At 70°C, the oil is mixed with the enzyme for 8 hours. When the reaction occurs, a sample of 200 to 250 mL of oil is extracted from the bottom of the container, and at the reaction temperature, the reaction product is filtered into a vacuum flask with a stirring rod through a vacuum Buchner funnel. The vacuum flask is placed on a hot plate, maintained at temperature with enough heat and stirred. The oil sample is analyzed using the technology described above. Unless otherwise stated, the dosage disclosed in the examples is the amount of fixed enzyme by weight of the oil.

[0226] Reaction completion is achieved when the physical properties of the oil do not change with increasing reaction time.Reaction completion is based on complete randomization of the fatty acids on the triglycerides and is determined by measuring the SFC, dropping point and carbon number of the triglycerides in the oil.

[0227] Table 3 below lists the profile of the oil produced in Experiment 1.

[0228] Table 3: 0.12 wt% at 70°C TL-IM (85:15 blend)

[0229]

[0230] Using a low dose (0.12wt%) TL-IM, the reaction did not reach completion after 8 h.

[0231] Test 2: 0.24 wt% at 70°C TL-IM; 85:15 blend

[0232] Apart from Experiment 2 was conducted using the same procedure as described in Experiment 1, except that the dosage of TL-IM was 0.24 wt%, and the reaction was allowed to proceed for 10 hours.

[0233] Table 4 below lists the profile of the oil produced in Experiment 2.

[0234] Table 4: 0.24 wt% at 70°C TL-IM (85:15 blend)

[0235]

[0236] At 70°C, the dosage was 0.24wt% TL-IM, the reaction did not reach completion after 10 h.

[0237] Test 3: 0.48 wt% at 70°C TL-IM; 85:15 blend

[0238] Apart from Run 3 was conducted using the same procedure as described for Run 1, except that the dosage of TL-IM was 0.48 wt%, and the reaction was allowed to proceed for 10 hours.

[0239] Table 5 below lists the profile of the oil produced in Experiment 3.

[0240] Table 5: 0.48 wt% Lipozyme TL-IM (85:15 blend) at 70°C

[0241]

[0242] At 70°C, the dosage was 0.48 wt% TL-IM, the reaction did not reach completion after 10 h.

[0243] Test 4: 0.72 wt% at 70°C TL-IM; 85:15 blend

[0244] Apart from Experiment 4 was conducted using the same procedure as described in Experiment 1, except that the dosage of TL-IM was 0.72 wt%, and the reaction was allowed to proceed for 10 hours. Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0245] Table 6 below lists the profile of the oil produced in Experiment 4.

[0246] Table 6: 0.72 wt% at 70°C TL-IM (85:15 blend)

[0247]

[0248] At 70°C, the dosage was 0.72wt / wt% TL-IM, the reaction reached completion after 8 hours.

[0249] Test 5: 0.72 wt% at 70°C TL-IM; 85:15 blend; enzyme reuse

[0250] In addition, the enzyme was 0.72 wt% of that used in the previous experiment 4. Experiment 5 was conducted using the same procedure as described for Experiment 4, except for TL-IM.

[0251] Table 7 below lists the profile of the oil produced in Experiment 5.

[0252] Table 7: 0.72 wt% at 70°C TL-IM (85:15 blend), enzyme reuse

[0253]

[0254] At 70°C, a dosage of 0.72 wt% of the TL-IM, the reaction did not reach completion after 10 h.

[0255] Test 6: 0.72 wt% dose of test enzyme I at 70°C; 85:15 blend

[0256] Experiment 6 was conducted using the same procedure as described in Experiment 1, except that the test enzyme I prepared as described in Example 2 was used at a dosage of 0.72 wt%. Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0257] Table 8 below lists the profile of the oil produced in Experiment 6.

[0258] Table 8: 0.72 wt% Test Enzyme I (85:15 blend) at 70°C

[0259]

[0260] At 70°C, using a dosage of 0.72 wt% of the test enzyme I, the reaction reached completion after 2 hours.

[0261] Test 7: 0.72 wt% test enzyme I at 70°C; 85:15 blend, enzyme reuse

[0262] Experiment 7 was conducted using the same procedure as described in Experiment 6, except that the enzyme was the 0.72 wt% test enzyme used previously in Experiment 6, and the reaction was allowed to proceed for 4 hours. Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0263] Table 9 below lists the profile of the oil produced in Experiment 7.

[0264] Table 9: Enzyme reuse at 0.72 wt% test enzyme I (85:15 blend) at 70°C

[0265]

[0266] At 70°C, using a dose of 0.72 wt% of a single-use test enzyme I (effective dose 0.36 wt%), the reaction reached completion after 2 hours.

[0267] Test 8: 0.72 wt% of test enzyme I at 70°C; 85:15 blend, enzyme reused twice

[0268] Trial 8 was conducted using the same procedure as described for Trial 7, except that the enzyme was 0.72 wt % of Test Enzyme I used previously in Trial 7 (previously used in Trials 6 and 7).

[0269] Table 10 below lists the profile of the oil produced in Experiment 8.

[0270] Table 10: Enzyme reuse at 70°C, 0.72 wt% test enzyme I (85:15 blend)

[0271]

[0272] At 70°C, using a dose of 0.72 wt% of the second re-used test enzyme I (effective dose 0.24 wt%), the reaction reached completion after 4 hours.

[0273] Test 9: 0.48 wt% at 80°C TL-IM; 85:15 blend

[0274] Experiment 9 was conducted using the same procedure as described for Experiment 3, except that the temperature was 80°C.

[0275] Table 11 below lists the profile of the oil produced in Experiment 9.

[0276] Table 11: 0.48 wt% at 80°C TL-IM (85:15 blend)

[0277]

[0278] At 80°C, the dosage was 0.48 wt% TL-IM, the reaction did not reach completion after 10 h.

[0279] Test 10: 0.48 wt% at 85°C TL-IM; 85:15 blend

[0280] Experiment 10 was conducted using the same procedure as described for Experiment 9, except that the temperature was 85°C.

[0281] Table 12 below lists the profile of the oil produced in Experiment 10.

[0282] Table 12: 0.48 wt% at 85°C TL-IM (85:15 blend)

[0283]

[0284] At 85°C, the dosage was 0.48 wt% TL-IM, the reaction did not reach completion after 10 hours. The sample collected at 8 hours reached all parameters, but then failed at 10 hours due to high MDP.

[0285] Test 11: 0.48 wt% at 90°C TL-IM; 85:15 blend

[0286] Experiment 11 was conducted using the same procedure as described for Experiment 9, except that the temperature was 90°C.

[0287] Table 13 below lists the profile of the oil produced in Experiment 11.

[0288] Table 13: 0.48 wt% at 90°C TL-IM (85:15 blend)

[0289]

[0290] At 90°C, the dosage was 0.48 wt% TL-IM, the reaction did not reach completion after 8 h.

[0291] Experiments 9-11 demonstrated that when the reaction temperature was raised above the temperature recommended by the manufacturer (ie, 70°C), the reaction could reach completion after a minimum of 8 hours.

[0292] Test 12: 0.72 wt% at 80°C TL-IM; 65:35 blend

[0293] Trial 12 was conducted using the same procedure as described in Trial 4, except that a 65:35 fractionated palm stearin / palm kernel oil blend was used and the temperature was 80°C.

[0294] Table 14 below lists the profile of the oil produced in Experiment 12.

[0295] Table 14 0.72wt% at 80°C TL-IM (65:35 blend)

[0296]

[0297] At 80°C, the dosage was 0.72wt%. TL-IM, the reaction reached completion after 8 hours.

[0298] Test 13: 0.24 wt% dose of test enzyme I at 80°C; 65:35 blend

[0299] Experiment 13 was conducted using the same procedure as described for Experiment 12, except that a dosage of 0.24 wt% of Test Enzyme I was used. Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0300] Table 15 below lists the profile of the oil produced in Experiment 13.

[0301] Table 15: 0.24 wt% Test Enzyme I (65:35 blend) at 80°C

[0302]

[0303] At 80°C, using a very low dose of 0.24 wt% of the test enzyme, the reaction reached completion after 10 hours.

[0304] Test 14: 0.24 wt% test enzyme I at 80°C; 65:35 blend, enzyme reuse

[0305] Experiment 14 was conducted using the same procedure as described for Experiment 13, except that the enzyme was Test Enzyme I at a 0.24 wt% dosage used previously for Experiment 13. Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0306] Table 16 below lists the profile of the oil produced in Experiment 14.

[0307] Table 16: Enzyme reuse at 80°C, 0.24 wt% test enzyme I (65:35 blend)

[0308]

[0309] At 80°C, using a single repeated use of the test enzyme at a dosage of 0.24 wt% (effective dosage 0.12 wt%), the reaction did not reach completion after 10 hours.

[0310] Test 15: 0.24 wt% test enzyme I at 80°C; 65:35 blend, enzyme reused twice

[0311] Experiment 15 was conducted using the same procedure as described for Experiment 13, except that the enzyme was Test Enzyme I (previously used in Experiments 13 and 14) at a 0.24 wt% dose used a second time in Experiment 14.

[0312] Table 17 below lists the profile of the oil produced in Experiment 15.

[0313] Table 17: 0.24 wt% test enzyme I (65:35 blend) at 80°C, enzyme secondary reuse

[0314]

[0315] At 80°C, using a second repeated use of the test enzyme I at a dosage of 0.24 wt% (effective dosage 0.08 wt%), the reaction did not reach completion after 10 hours.

[0316] Test 16: 0.24 wt% dose of test enzyme I at 80°C; 65:35 blend

[0317] Experiment 16 was performed using the same procedure as described for Experiment 13.

[0318] Table 18 below lists the profile of the oil produced in Experiment 16.

[0319] Table 18: 0.24 wt% Test Enzyme I (65:35 blend) at 80°C

[0320]

[0321] At 80°C, using a dosage of 0.24 wt% of the test enzyme I, the reaction reached completion after 6 hours.

[0322] Test 17: 0.24 wt% dose of test enzyme I at 85°C; 65:35 blend

[0323] Experiment 17 was conducted using the same procedure as described for Experiment 13, except that the temperature was 85°C.

[0324] Table 19 below lists the profile of the oil produced in Experiment 17.

[0325] Table 19: 0.24 wt% Test Enzyme I (65:35 blend) at 85°C

[0326]

[0327]

[0328] At 85°C, using a dosage of 0.24 wt% of the test enzyme I, the reaction reached completion after 6 hours.

[0329] Test 18: 0.24 wt% dose of test enzyme I at 90°C; 65:35 blend

[0330] Experiment 18 was conducted using the same procedure as described for Experiment 13, except that the temperature was 90°C.

[0331] Table 20 below lists the profile of the oil produced in Experiment 18.

[0332] Table 20: 0.24 wt% Test Enzyme I (65:35 blend) at 90°C

[0333]

[0334]

[0335] At 90°C, using a dosage of 0.24 wt% of the test enzyme I, the reaction reached completion after 6 hours.

[0336] Test 19: 0.24 wt% dose of test enzyme I at 95°C; 65:35 blend

[0337] Experiment 19 was conducted using the same procedure as described for Experiment 13, except that the temperature was 95°C.

[0338] Table 21 below lists the profile of the oil produced in Experiment 19.

[0339] Table 21: 0.24 wt% Test Enzyme I (65:35 blend) at 95°C

[0340]

[0341]

[0342] At 95°C, the reaction reached completion after 10 hours using a dosage of 0.24 wt% of the test enzyme I. The reduction in reaction rate compared to experiments 16-18 is a result of enzyme denaturation.

[0343] Test 20: 0.36 wt% dose of test enzyme I at 80°C; 65:35 blend

[0344] Experiment 20 was performed using the same procedure as described for Experiment 13, except that 0.36 wt% of Test Enzyme I was used.

[0345] Table 22 below lists the profile of the oil produced in Run 20.

[0346] Table 22: 0.36 wt% Test Enzyme I (65:35 blend) at 80°C

[0347]

[0348]

[0349] At 80°C, using a low dose of 0.36 wt% of test enzyme I, the reaction reached completion after 4 hours.

[0350] Test 21: 0.48 wt% dose of test enzyme I at 80°C; 65:35 blend

[0351] Experiment 21 was carried out using the same procedure as described for Experiment 13, except that 0.48 wt% of Test Enzyme I was used. Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0352] Table 23 below lists the profile of the oil produced in Experiment 21.

[0353] Table 23: 0.48 wt% Test Enzyme I (65:35 blend) at 80°C

[0354]

[0355]

[0356] At 80°C, using a low dose of 0.48 wt% of the test enzyme, the reaction reached completion after 2 hours.

[0357] Test 22: 0.48 wt% test enzyme at 80°C; 65:35 blend, enzyme reuse

[0358] Experiment 22 was conducted using the same procedure as described for Experiment 21, except that the enzyme was the 0.48 wt % test enzyme used previously in Experiment 21 plus an additional 33 wt % of fresh (unused) test enzyme (based on the weight of the test enzyme used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0359] Table 24 below lists the profile of the oil produced in Experiment 22.

[0360] Table 24: Enzyme reuse at 80°C, 0.48 wt% test enzyme I (65:35 blend)

[0361]

[0362]

[0363] At 80°C, using a dose of 0.48 wt% of a single-use test enzyme I in combination with an additional 33% of fresh (unused) test enzyme (effective dose 0.32 wt%), the reaction reached completion after 2 hours.

[0364] Test 23: 0.48 wt% of test enzyme I at 80°C; 65:35 blend, enzyme reused 2 times

[0365] Test 23 was conducted using the same procedure as described for Test 21, except that the enzyme was 0.48 wt % Test Enzyme I (used in Tests 21 and 22) previously used a second time in Test 22 plus an additional 25 wt % of fresh (unused) Test Enzyme I (based on the weight of Test Enzyme I previously used). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0366] Table 25 below lists the profile of the oil produced in Experiment 23.

[0367] Table 25: Enzyme reuse at 80°C, 0.48 wt% test enzyme I (65:35 blend)

[0368]

[0369]

[0370] At 80°C, using a dosage of 0.48 wt% of the second reused test enzyme I in combination with an additional 25% fresh (unused) test enzyme I (based on the weight of the previously used test enzyme I) (effective dosage of 0.27 wt%), the reaction reached completion after 2 hours.

[0371] Test 24: 0.48 wt% of test enzyme I at 80°C; 65:35 blend, enzyme reused 3 times

[0372] Test 24 was conducted using the same procedure as described for Test 21, except that the enzyme was 0.48 wt % Test Enzyme I used three times previously in Test 23 (for Tests 21, 22, and 23) plus an additional 20 wt % of fresh (unused) Test Enzyme I (based on the weight of Test Enzyme I used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0373] Table 26 below lists the profile of the oil produced in Experiment 24.

[0374] Table 26: 0.48 wt% test enzyme I (65:35 blend) at 80°C, enzyme reused 3 times

[0375]

[0376]

[0377] At 80°C, the reaction reached completion after 2 hours using a combination of three repeated uses of the test enzyme I at a dosage of 0.48 wt% and an additional 20% fresh (unused) test enzyme I (based on the weight of the previously used test enzyme I) (effective dosage 0.24 wt%).

[0378] Test 25: 0.48 wt% of test enzyme I at 80°C; 65:35 blend, enzyme reused 4 times

[0379] Experiment 25 was conducted using the same procedure as described for Experiment 21, except that the enzyme was 0.48 wt % Test Enzyme I used four times previously in Experiment 24 (for Experiments 21, 22, 23 and 24) plus an additional 17 wt % fresh (unused) Test Enzyme I (based on the weight of Test Enzyme I previously used).

[0380] Table 27 below lists the profile of the oil produced in Experiment 25.

[0381] Table 27: 0.48 wt% test enzyme I (65:35 blend) at 80°C, enzyme reused 4 times

[0382]

[0383] At 80°C, using a dosage of 0.48 wt% of the test enzyme I used four times previously in combination with an additional 17% of fresh (unused) test enzyme I (effective dosage of 0.23 wt%), the reaction reached completion after 2 hours.

[0384] The second immobilization support was used for the next series of exemplary test enzymes II.

[0385] Test 26: 0.48 wt% dose of test enzyme II at 80°C; 65:35 blend

[0386] Experiment 26 was performed using the same procedure as described for Experiment 21 except that the dosage was 0.48 wt% and Test Enzyme II was utilized.

[0387] Table 28 below lists the profile of the oil produced in Experiment 26.

[0388] Table 28: 0.48 wt% Test Enzyme II (65:35 blend) at 80°C

[0389]

[0390] At 80°C, using a dosage of 0.48 wt% of the test enzyme II, the reaction did not reach completion after only two hours.

[0391] Test 27: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reuse

[0392] Experiment 27 was conducted using the same procedure as described for Experiment 26, except that the enzyme was the 0.48 wt % test enzyme used previously in Experiment 26 plus an additional 33 wt % of fresh (unused) test enzyme II (based on the weight of the test enzyme used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0393] Table 29 below lists the profile of the oil produced in Experiment 27.

[0394] Table 29: Enzyme reuse at 80°C, 0.48 wt% test enzyme II (65:35 blend)

[0395]

[0396] At 80°C, using a dose of 0.48 wt% of a single reused test enzyme II in combination with an additional 33% of fresh (unused) test enzyme II (effective dose 0.32 wt%), the reaction reached completion after 2 hours.

[0397] Test 28: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 2 times

[0398] Test 28 was conducted using the same procedure as described for Test 27, except that the enzyme was 0.48 wt % of the Test Enzyme II previously used a second time in Test 27 (for Tests 26 and 27) plus an additional 25 wt % of fresh (unused) Test Enzyme II (based on the weight of the Test Enzyme II previously used). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0399] Table 30 below lists the profile of the oil produced in Run 28.

[0400] Table 30: 0.48 wt% test enzyme II (65:35 blend) at 80°C, enzyme secondary reuse

[0401]

[0402] At 80°C, using a dosage of 0.48 wt% of the second reused test enzyme II in combination with an additional 25% fresh (unused) test enzyme II (based on the weight of the previously used test enzyme II) (effective dosage of 0.27 wt%), the reaction reached completion after 2 hours.

[0403] Test 29: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 3 times

[0404] Run 29 was conducted using the same procedure as described for Run 26, except that the enzyme was 0.48 wt % of Test Enzyme II used three times previously in Run 30 (for Runs 26, 27, and 28) plus an additional 20 wt % of fresh (unused) Test Enzyme II (based on the weight of Test Enzyme II used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0405] Table 31 below lists the profile of the oil produced in Experiment 29.

[0406] Table 31: 0.48 wt% test enzyme II (65:35 blend) at 80°C, enzyme reused 3 times

[0407]

[0408] At 80°C, the reaction reached completion after 2 hours using a combination of three repeated uses of the test enzyme II at a dosage of 0.48 wt% and an additional 20% fresh (unused) test enzyme II (based on the weight of the previously used test enzyme II) (effective dosage of 0.24 wt%).

[0409] Test 30: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 4 times

[0410] Experiment 30 was conducted using the same procedure as described for Experiment 26, except that the enzyme was 0.48 wt % of the test enzyme II used four times previously in Experiment 31 (used in Experiments 26, 27, 28 and 29) plus an additional 17 wt % of fresh (unused) test enzyme II (based on the weight of the test enzyme II previously used).

[0411] Table 32 below lists the profile of the oil produced in Experiment 30.

[0412] Table 32: 0.48 wt% test enzyme II (65:35 blend) at 80°C, enzyme reused 4 times

[0413]

[0414] At 80°C, using a dosage of 0.48 wt% of the test enzyme II from four previous uses in combination with an additional 17% of fresh (unused) test enzyme II (effective dosage of 0.23 wt%), the reaction reached completion after 2 hours.

[0415] Test 31: 0.48 wt% dose of test enzyme II at 80°C; 65:35 blend

[0416] Experiment 31 was conducted using the same procedure as described for Experiment 26, except that sampling began at 0.5 hours. Samples were collected at 0.5 hours, 1.0 hours, 1.5 hours, 2.0 hours, and 4.0 hours.

[0417] Table 33 below lists the profile of the oil produced in Experiment 31.

[0418] Table 33: 0.48 wt% Test Enzyme II (65:35 blend) at 80°C

[0419]

[0420] At 80°C, using a dosage of 0.48 wt% of the test enzyme II, the reaction did not reach completion after only one and a half (1.5) hours.

[0421] Test 32: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reuse

[0422] Test 32 was conducted using the same procedure as described for Test 31, except that the enzyme was the 0.48 wt % test enzyme used previously in Test 32 plus an additional 33 wt % of fresh (unused) test enzyme II (based on the weight of the test enzyme used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0423] Table 34 below lists the profile of the oil produced in Experiment 32.

[0424] Table 34: Enzyme reuse at 80°C, 0.48 wt% test enzyme II (65:35 blend)

[0425]

[0426]

[0427] At 80°C, using a dose of 0.48 wt% of a single reused test enzyme II in combination with an additional 33% fresh (unused) test enzyme II (effective dose of 0.32 wt%), the reaction reached completion after one and a half (1.5) hours.

[0428] Test 33: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 2 times

[0429] Test 33 was conducted using the same procedure as described for Test 31, except that the enzyme was the 0.48 wt % test enzyme used previously in Test 32 (the enzyme was used in Tests 31 and 32) plus an additional 25 wt % of fresh (unused) test enzyme II (based on the weight of the test enzyme used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0430] Table 35 below lists the profile of the oil produced in Experiment 33.

[0431] Table 35: Enzyme reuse at 80°C, 0.48 wt% test enzyme II (65:35 blend)

[0432]

[0433]

[0434] At 80°C, using a dosage of 0.48 wt% of the second reused test enzyme II in combination with an additional 25% fresh (unused) test enzyme II (effective dosage of 0.27 wt%), the reaction reached completion after one and a half (1.5) hours.

[0435] Test 34: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 3 times

[0436] Test 34 was conducted using the same procedure as described for Test 31, except that the enzyme was the 0.48 wt% test enzyme used previously in Test 33 (the enzyme was used in Tests 31, 32, and 33) plus an additional 20 wt% of fresh (unused) test enzyme II (based on the weight of the test enzyme used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0437] Table 36 below lists the profile of the oil produced in Experiment 34.

[0438] Table 36: 0.48 wt% test enzyme II (65:35 blend) at 80°C, enzyme reused 3 times

[0439]

[0440]

[0441] At 80°C, using three replicates of Test Enzyme II at a dosage of 0.48 wt% in combination with an additional 20% fresh (unused) Test Enzyme II (effective dosage of 0.24 wt%), the reaction reached completion after one and a half (1.5) hours.

[0442] Test 35: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 4 times

[0443] Test 35 was conducted using the same procedure as described for Test 31, except that the enzyme was the 0.48 wt% test enzyme used previously in Test 34 (the enzyme was used in Tests 31, 32, 33, and 34) plus an additional 17 wt% of fresh (unused) test enzyme II (based on the weight of the test enzyme used previously). Once the immobilized enzyme was removed from the oil, it was stored in a sealed container for reuse.

[0444] Table 37 below lists the profile of the oil produced in Experiment 35.

[0445] Table 37: 0.48 wt% test enzyme II (65:35 blend) at 80°C, enzyme reused 4 times

[0446]

[0447]

[0448] At 80°C, using four repeated uses of Test Enzyme II at a dosage of 0.48 wt% in combination with an additional 17% fresh (unused) Test Enzyme II (effective dosage of 0.23 wt%), the reaction reached completion after one and a half (1.5) hours.

[0449] Test 36: 0.48 wt% test enzyme II at 80°C; 65:35 blend, enzyme reused 5 times

[0450] Run 36 was performed using the same procedure as described for Run 31, except that the enzyme was 0.48 wt% of the test enzyme previously used in Run 35 (the enzyme was used in Runs 31, 32, 33, 34, and 35). No additional fresh enzyme was used.

[0451] Table 38 below lists the profile of the oil produced in Experiment 36.

[0452] Table 38: 0.48 wt% test enzyme II (65:35 blend) at 80°C, enzyme reused 5 times

[0453]

[0454]

[0455] At 80°C, using five repeated uses of the test enzyme II at a dosage of 0.48 wt% (effective dosage 0.18 wt%), the reaction reached completion after two hours.

[0456] Summary of results

[0457] The results of Experiments 1-36 are summarized in Table 39 below.

[0458] Table 39

[0459]

[0460]

[0461]

[0462] Example 2: Preparation of Test Enzyme I

[0463] The carrier used to prepare the test enzyme I was HybCPG VBC (Fe 3+ ), wherein the test enzyme was prepared according to the procedure described in WO 2015 / 115993. TL100L fixed to HybCPG VBC(Fe 3+ ) on the carrier.

[0464] First, an enzyme solution was prepared, which contained 40 vol% TL 100L, and 3.84g / L (20mM) citric acid and 0.01g / L (0.05mM) calcium chloride hexahydrate. Add citric acid and calcium chloride hexahydrate as pre-made stock solutions to 40vol% TL 100L solution, in which the concentration of citric acid was 250mM, the pH was adjusted to pH 7, and calcium chloride was 200mM. The pH of the final TLL enzyme solution for immobilization was 6.6. 1L of enzyme solution was used with 25g of HybCPG VBC carrier

[0465] The HybCPG VBC carrier and TLL enzyme solution were mixed on a drum mixer at 60 rpm for 1.5 hours, and then the immobilized catalyst (on HybCPG VBC) was TL 100L) precipitation, and the supernatant is decanted. The fixed catalyst is washed 3 times with a buffer solution (160ml per 25g of carrier) containing 5.78g / L (75mM) ammonium acetate, 23.8g / L (100mM) 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) and 2.19g / L (10mM) calcium chloride hexahydrate (pH 7). For each washing step, the fixed catalyst is mixed with the washing solution on a drum mixer at 60rpm for 1 minute, precipitated, and then the supernatant is removed by decanting. Then, the fixed catalyst is transferred to a borosilicate glass 3.3 filter funnel (porosity is 3 levels) using washing solution (70ml per 25g of carrier), and is dried for 1.5 hours by removing liquid by means of a vacuum pump (150mbar below atmospheric pressure). Then, the carrier is transferred to a crystallization tray, and it is subjected to vacuum (980mbar below atmospheric pressure) for 48 hours.

[0466] In the activity assay based on tributyrin hydrolysis using pH constant instrument, by comparing the activity of TLL enzyme solution before and after fixing, the amount of TLL fixed on EziG has been determined.Reaction mixture is 5ml tributyrin and 45ml buffer (2.09g / 1,10mM, 3-morpholinopropane-1-sulfonic acid; 43.8g / l, 0.2M, calcium chloride hexahydrate; and 20g / l gum arabic (gum arabic), pH 7.5) emulsion and.By measuring the amount of the alkaline solution (20g / l, 0.5M, sodium hydroxide) added per minute and the volume of TLL enzyme solution, determine that activity continues for 4 minutes at 40 ℃ and pH 7.5. The activity of TLL in the solution phase has previously been determined to be 5600 μmol per minute per mg of enzyme at 25°C (M.Martinelle, M.Holmquist, K.Hult, Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1995, 1258, 272-276. https: / / doi.org / 10.1016 / 0005-2760(95)00131-U), and was used to quantify the amount of immobilized TLL on the carrier. Using the protocol described herein, the amount of TLL immobilized on the carrier was 0.08 g per gram of carrier. However, depending on the details of the preparation, the amount of TLL immobilized on the carrier can be adjusted from 0.08 g per gram of carrier to 0.16 g.

[0467] The test enzyme I used in experiments 6, 7, 8, 13 and 15 had 15.9% w / w bound enzyme (enzyme loading). The test enzyme I used in experiments 16-25 contained 8.6% w / w bound enzyme (enzyme loading).

[0468] Example 3: Preparation of Test Enzyme II

[0469] The silica carrier used in the preparation was Cariact Q30 purchased from Fuji Silicon Chemical Co., Ltd. (Cariact Q30 has a particle size of 70-150 μm, a pore size of 30 nm, and a surface area of ​​99 m 2). The silica support (5 g) was transferred to a jacketed glass reactor with an overhead stirrer, and toluene (100 mL) was added. The surface of the silica support was amino-functionalized using 15 mL of (3-aminopropyl) trimethoxysilane, which was added to the reactor and incubated for 22 hours (250-290 rpm, 80° C.). The resulting solid was filtered under vacuum, washed with toluene (2×200 mL), and then washed with EtOH 99.7% (2×200 mL). After the washing step, the solid was filtered under vacuum using a glass filter funnel for 60 minutes. The amino-functionalized support was transferred to a crystallization tray and dried overnight at 120° C. under vacuum.

[0470] Thermomyces lanuginosus lipase (TLL) was prepared by diluting the commercial enzyme solution (60% v / v) into deionized water (final solution pH 6.7). TL 100L of solution was used for fixation. The diluted enzyme solution (250mL) was transferred to a 500mL Duran bottle containing 50g of carrier (CPS Q30 functionalized with (3-aminopropyl) trimethoxysilane). The sample was incubated on a drum mixer for 1.5 hours (70rpm, ambient temperature). After fixation, the supernatant was removed and the carrier was washed with a solution containing 50mM HEPES, 50mM ammonium acetate, 10mM CaCl2, pH 7 solution (1×250mL), and then further washed with a solution containing 50mM HEPES, 50mM ammonium acetate, 10mM CaCl2, 130mM sucrose, pH 7 solution (1×250mL). For each washing step, the immobilized catalyst was mixed with the washing solution on a drum mixer for 3 minutes (70rpm, ambient temperature). The catalyst was filtered on a borosilicate glass 3.3 filter funnel under vacuum to remove most of the wash solution (6 min), transferred to a 1 L glass flask and dried on a rotary evaporator for 115-120 min (40 mbar, 40° C.).

[0471] The amount of TLL immobilized on the support was typically 0.05 g per gram of support (5% enzyme loading), determined as described above for Test Enzyme I.

[0472] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any device or system and performing any incorporated method. The patentable scope of the invention is defined by the claims, and may include other examples that occur to a person skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that are not substantially different from the literal language of the claims.

Claims

1. A process for batch enzymatic treatment of a plurality of lipid-containing compositions, the process comprising: providing a lipid-containing composition; contacting the lipid-containing composition with an immobilized enzyme material comprising a carrier and at least one enzyme immobilized on the carrier, wherein the amount of the immobilized enzyme material is about 0.72% or less (based on the weight of the lipid-containing composition); mixing the lipid-containing composition with the immobilized enzyme material to form a first enzymatically treated composition, wherein the mixing occurs for about 10 hours or less; separating the immobilized enzyme material from the first enzymatically treated composition; contacting the immobilized enzyme material with at least one additional lipid-containing composition; and The at least one additional lipid-containing composition is mixed with the immobilized enzyme material to form a second enzymatically treated composition.

2. The process of claim 1, further comprising separating the immobilized enzyme material from the second enzymatically treated composition.

3. The process according to any one of claims 1 to 2, wherein the amount of immobilized enzyme material is from about 0.15% to about 0.72% (by weight of the lipid-containing composition).

4. The process of claim 3, wherein the amount of the immobilized enzyme material is from about 0.18% to about 0.72% (by weight of the lipid-containing composition).

5. The process of claim 4, wherein the amount of immobilized enzyme material is from about 0.18% to about 0.5% (by weight of the lipid-containing composition).

6. The process of any one of claims 1 to 5, wherein the mixing occurs for about 1.5 hours to about 10 hours.

7. The process according to any one of claims 1 to 6, wherein the lipid-containing composition is heated to a temperature of about 70°C to about 95°C.

8. The process according to any one of claims 1 to 7, wherein the enzymatic treatment is selected from the group consisting of transesterification, intraesterification, alcoholysis, acidolysis, glycerolysis, transesterification and combinations thereof.

9. The process of any one of claims 1 to 8, wherein the enzyme is derived from Achromobacter, Alcaligenes, Aspergillus, Bacillus, Burkholderia, Candida, Chromobacterium, Corynebacterium, Fusarium, Geotrichum, Humicola, Humicola, Mucor, Penicillium, Pseudomonas, Rhizomucor, Rhizopus, Staphylococcus, Thermomyces or Torulopsis.

10. The process of claim 9, wherein the enzyme is selected from the group consisting of Aspergillus niger Mucor mihei, Mucor javanicus Pseudomonas Fuorescens, Rhizopus delemar, Burkholderia cepacia Candida cylindracea, Candida Antarctica Candida rugosa Fusarium solani Penicillium cyclopium, Rhizomucor miehei and Thermomyces lanuginosus.

11. The process of claim 10, wherein the enzyme is Thermomyces lanuginosus.

12. The process according to any one of claims 1 to 11, wherein the lipid-containing composition comprises an oil or fat selected from the group consisting of canola oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, hazelnut oil, hempseed oil, linseed oil, meadowfoam oil, olive oil, palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, soybean oil, sunflower oil, tall oil, tsubaki oil, kohlrabi ... oil), various "natural" oils with fatty acid compositions altered by genetically modified organisms (GMOs) or traditional "breeding," such as high oleic or low linolenic, low saturated oils (high oleic canola, low linolenic soybean, or high stearic sunflower), vegetable oils, menhaden oil, wax fish oil, cod liver oil, orange crude oil, sardine oil, menhaden oil, lard, tallow, algae oil, fish oil, fats of animal origin, waste cooking oils, brown grease, oil triglycerides derived from inedible plant sources, partial glycerides and free fatty acids derived from these oils, and combinations or blends thereof.

13. The process according to any one of claims 1 to 12, wherein the enzymatic treatment is carried out in a stirred tank reactor operated in batch mode.

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