Radial partition water activity buffer type immobilized lipase and preparation method and application thereof

Abstract

This invention relates to the field of immobilized enzymes and functional oil biocatalysis, and discloses a radially partitioned water-activity buffered immobilized lipase, its preparation method, and its applications. The immobilized lipase comprises a carbon core and an outer radially partitioned silica shell. From the inside out, the shell sequentially comprises a water-activity buffer zone, a lipase immobilization zone, and a substrate diffusion zone. During preparation, an inner porous silica layer is first formed, and trehalose, sorbitol, and oligosilicic acid are introduced into the inner pores and gelled in situ. Subsequently, the lipase immobilization zone and substrate diffusion zone are sequentially constructed epitaxially. The lipase immobilization zone has hydrophobic adsorption groups and flexible spacer arms containing epoxy end groups. This structure is used for the glycerolization of vegetable oils to prepare diacylglycerol oils.

Description

Technical Field

[0001] This invention belongs to the technical field of immobilized enzymes, biocatalytic oil modification and functional oil preparation. Specifically, it relates to an immobilized lipase in which a water activity buffer zone, a lipase immobilization zone and a substrate diffusion zone are sequentially constructed radially inside the immobilized lipase particle, the method of sequential construction thereof, and its application in the preparation of diacylglycerol oil by glycerolysis of vegetable oils. Background Technology

[0002] Lipases can catalyze reactions such as glycerol hydrolysis, esterification, and transesterification in non-aqueous or low-aqueous systems. Lipases require a certain amount of bound water to maintain their catalytic conformation; too little water restricts protein conformational movement, while too much water may enhance hydrolytic competition and alter the distribution of polar components such as glycerol near the immobilized carrier and enzyme interface. Therefore, in low-aqueous multiphase systems, describing the hydration state of immobilized lipases by total system water content has limitations. Water activity and the distribution of water among the enzyme, carrier, and bulk phase are equally important and have a significant impact on the efficiency of diacylglycerol catalytic synthesis.

[0003] Existing technologies have proposed controlling the moisture state in non-aqueous enzymatic reactions by adding water-absorbing materials or external moisture regulating components to the reactor. WO1982003873A1 relates to the control of water activity in non-aqueous enzymatic reactions; US4818695A relates to the control of water content in immobilized lipases; EP2085469A1 relates to the combination of lipase and boehmite; WO2012050422A1 relates to the use of immobilized lipases for the glycerol hydrolysis of oils and the control of glycerol addition and moisture.

[0004] Glycerolysis of vegetable oils itself does not produce water; the water in the system mainly comes from the oil, glycerol, immobilized enzymes, water adsorbed by the carrier, and moisture absorbed from the environment. Glycerol has strong hydrophilicity and high viscosity, which can easily alter the local polarity environment near the enzyme. Therefore, spatial specialization is needed for water activity buffering, lipase immobilization, and substrate mass transfer during particle formation. Summary of the Invention

[0005] 1. Technical problems to be solved This invention aims to address the problem that the water activity of the immobilized lipase microenvironment during the preparation of diacylglycerol from triacylglycerol is easily affected by the water content of the raw materials, glycerol distribution, and the adsorption behavior of the carrier. It establishes a local water activity buffer structure inside the particle and reduces the mass transfer resistance of oil macromolecules through the outer macroporous channels, thereby improving the stability of continuous reaction and the selectivity of diacylglycerol.

[0006] 2. Technical Solution This invention uses carbon core@radial gradient pore silica composite particles as a carrier to construct a water activity buffer zone, a lipase immobilization zone, and a substrate diffusion zone sequentially from the inside to the outside along the radial direction. The water activity buffer zone has a 2-6 nm micropore structure, within which a hydrophilic silica-trehalose-sorbitol composite buffer phase is confined; the lipase immobilization zone has a 10-25 nm mesopore structure, with a hydrophobic adsorption interface formed by C8 alkyl groups, and secondary immobilization of the lipase is achieved through a polyether flexible spacer arm containing epoxy end groups; the substrate diffusion zone has 30-80 nm open channels.

[0007] After completing the water activity buffer zone, a mesoporous lipase immobilization zone is formed on its outer side. Hydrophobic adsorption groups and flexible organic spacers containing reactive end groups are introduced onto the pore surface of the lipase immobilization zone, allowing the lipase to first undergo hydrophobic interfacial adsorption, followed by secondary immobilization through the reactive end groups. Subsequently, an open substrate diffusion zone with a larger pore size is formed on the outer side of the lipase immobilization zone.

[0008] Preferably, the average pore size of the inner water activity buffer zone is 2–6 nm, the average pore size of the middle lipase immobilization zone is 10–25 nm, and the average pore size of the outer substrate diffusion zone is 30–80 nm. Solvent extraction is preferably used to remove the template agent used in subsequent layering.

[0009] To further reduce the migration of buffer components during subsequent layering and operation, the degree of condensation of the silica gel network within the pores can be increased; alternatively, some polyol groups can participate in the formation of the Si-O-Si network through hydrolyzable silicon-containing organic precursors.

[0010] 3. Beneficial effects.

[0011] (1) By shortening the local water migration distance by setting the inner buffer phase and the middle enzyme immobilization zone adjacent to each other, the water activity regulation occurs inside the immobilized enzyme particles, rather than relying on the strong desiccant added independently in the reactor. (2) By separating the hydrophilic buffer phase from the hydrophobic enzyme immobilization interface, the conditions for lipase hydration and hydrophobic interface activation can be maintained simultaneously, reducing the excessive accumulation of glycerol or water on the enzyme surface. (3) The open pores of the outer layer of 30-80 nm provide mass transfer channels for triacylglycerol, diacylglycerol and monoacylglycerol, avoiding diffusion resistance caused by overall hydrophilicity and small pore structure; (4) Applicable to batch and fixed-bed continuous glycerol hydrolysis. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the structure of the radially partitioned water activity buffer type immobilized lipase of the present invention.

[0013] 11—Carbon core; 12—Water activity buffer zone; 13—Lipase immobilization zone; 14—Substrate diffusion zone; 15—Lipase; 16—Water activity buffer component. Detailed Implementation

[0014] The following embodiments are used to illustrate the technical solution of the present invention and do not constitute a limitation on the scope of protection. Unless otherwise stated, all percentage contents are by mass. The listed process parameters can be adjusted within the range described in the present invention according to the carrier pore volume, shell thickness, and equipment dimensions.

[0015] Example 1: Construction of the inner water activity buffer zone (1) Carbon core pretreatment: Take 10.0 g of phenolic resin-based spherical carbon particles with a particle size of 150-300 μm, wash with 1.0 mol / L hydrochloric acid for 2 h, wash with water until neutral; then oxidize with 20% nitric acid at 75℃ for 2 h, wash and vacuum dry at 80℃.

[0016] (2) Formation of inner porous silica: Using tetraethyl orthosilicate as the silicon source, an inner porous silica layer with an average pore size of 2-6 nm is formed on the surface of the carbon core. The template agent is removed by solvent extraction to obtain an intermediate containing only the carbon core and the inner porous silica layer.

[0017] (3) Preparation of oligosilicic acid impregnation solution: Tetraethyl orthosilicate is pre-hydrolyzed to form oligosilicic acid, trehalose and sorbitol are added, so that the content of trehalose and sorbitol in the impregnation solution is 2.5-3.5% by mass, the content of oligosilicic acid based on silicon dioxide is 2-4% by mass, and the pH is adjusted to 4.5-5.5.

[0018] (4) Pore confinement: The intermediate was vacuum dried at 60°C for 4 h. Based on the available pore volume of the inner layer, an impregnation solution equivalent to 70-95% of the available pore volume of the inner layer was added in a single step. Vacuum impregnation was carried out at -0.06 to -0.09 MPa for 10-20 min. After restoring to normal pressure, the mixture was allowed to stand for 20 min to remove excess liquid from the outer surface of the particles. The oligomeric silicate was then condensed and gelled at 35-45°C and aged at 50-70°C for 2-6 h. The impregnation and gelation were repeated 2-3 times to obtain a water activity buffer containing trehalose and sorbitol.

[0019] Example 2: Sequential epitaxial construction of lipase immobilization region and substrate diffusion region A mesoporous silica layer with an average pore size of 10–25 nm was formed on the outer side of the intermediate obtained in Example 1. CTAB was used as a template agent, and the pore size was adjusted by a pore-expanding agent. After the mesoporous layer was formed, C8 alkyl hydrophobic groups and polyether flexible spacer arms containing epoxy end groups were introduced onto its pore surface through copolymerization and post-grafting.

[0020] An open macroporous silica layer with an average pore size of 30–80 nm is further epitaxially formed outside the mesoporous lipase immobilization region to obtain a radially partitioned shell. The template agent is then removed by extraction.

[0021] Example 3: Lipase immobilization of Thermomyces lanuginosus A Thermomyces lanuginosus lipase solution was prepared using 20 mmol / L phosphate buffer (pH 7.5), with a protein concentration of 8–12 mg / mL. The carrier obtained in Example 2 was contacted with the enzyme solution and the mixture was shaken at 20–22°C for 10–14 h to allow the lipase to enter the lipase immobilization region through the outer open pores and adsorb at the C8 hydrophobic interface. After filtration, the mixture was transferred to pH 8.0 buffer and reacted at 24–26°C for 2–4 h to allow secondary immobilization of the adsorbed lipase with the adjacent epoxy end groups. After washing, the mixture was equilibrated in a humidified environment at 25°C to achieve the set water activity and total water content of the immobilized lipase.

[0022] Example 4: Evaluation of glycerol hydrolysis catalytic performance Refined vegetable oil was vacuum dehydrated at 80℃ for 2 h, and food-grade glycerol was vacuum dehydrated at 85℃ for 1 h. The reaction substrate was prepared at a triacylglycerol to glycerol molar ratio of 1:0.60. The immobilized lipase to be tested was pre-equilibrated at 25℃, and its water activity, total water content, and immobilized protein loading were measured. The catalyst dosage was standardized based on the amount of immobilized protein.

[0023] The reaction temperature was 60℃. The total amount of glycerol was added in three portions: 40% at 0 h, 30% at 2 h, and 30% at 4 h. The initial DAG generation rate was fitted to the linear region from 0 to 60 min and normalized to the fixed protein amount.

[0024] Comparative Example Comparative Example 1: The same radially partitioned silica carrier as in Example 2 and the same lipase immobilization method as in Example 3 were used, but the trehalose, sorbitol, and oligosilicic acid composite buffer phase was not introduced into the inner layer. Humidity control was employed to maintain an initial total moisture content similar to that of the immobilized lipase obtained in Example 3.

[0025] Comparative Example 2: Using the unbuffered immobilized lipase of Comparative Example 1, boehmite particles of equivalent mass to the composite buffer phase of Example 1 were added to the reaction system. The boehmite was pre-equilibrated in a specified relative humidity environment at 25°C to ensure it was not added in a completely dry state; the remaining reaction conditions, the amount of immobilized protein added, and the initial total water content remained consistent with those of the Example.

[0026] Table 4 compares the catalytic effects of immobilized lipases in Example 4 with those in the comparative examples. Group Initial water activity at 25°C (aw) Total moisture content / % 6 h DAG / % FFA / % 72 h activity retention / % Activity retention rate after 168 h / % Example 4 0.38 3.6 52 0.7 94 89 Comparative Example 1 0.38 3.6 44 1.3 83 74 Comparative Example 2 0.38 3.6 48 0.9 89 83 As can be seen from the table, the immobilized lipase prepared by this invention has higher DAG content and catalyst activity retention, as well as lower fatty acid FFA content.

[0027] Detection and Evaluation Methods (1) Water activity: measured at a constant temperature of 25°C using a water activity meter calibrated with standard salt.

[0028] (2) Moisture content: determined by Karl Fischer method.

[0029] (3) Immobilized protein load: The protein concentration in the enzyme solution and washing solution before and after immobilization was measured, and the immobilized protein load was calculated according to the law of conservation of mass.

[0030] (4) Glyceryl ester composition: DAG and FFA were determined by liquid chromatography.

[0031] (5) Stability: The DAG generation rate was determined at 72 h and 168 h using the same standardized method as the initial value; the activity retention rate was calculated as the ratio of the DAG generation rate at the corresponding time point to the initial DAG generation rate.

[0032] Industrial applicability This invention integrates water activity buffering function, hydrophobic lipase immobilization function and oil macromolecule diffusion function into the same porous particle, which can be used to prepare diacylglycerol oil by glycerolysis of vegetable oil, and is suitable for batch reactors, fixed beds, radial flow fixed beds or circulating fixed beds.

Claims

1. A radially partitioned water activity buffered immobilized lipase, characterized in that, It includes a carbon core and a silica shell covering the outside of the carbon core; The silica shell includes, in a radial direction from the carbon core to the outer surface of the particle, a water activity buffer zone, a lipase immobilization zone, and a substrate diffusion zone that are interconnected by channels. The average pore size of the water activity buffer zone is smaller than the average pore size of the lipase immobilization zone, and the average pore size of the lipase immobilization zone is smaller than the average pore size of the substrate diffusion zone. The water activity buffer zone contains trehalose and sorbitol confined by a silica gel network; The pore surface of the lipase immobilization region is provided with hydrophobic adsorption groups and flexible organic spacer arms containing reactive end groups. The lipase is first adsorbed at the interface through the hydrophobic adsorption groups, and then immobilized again through the reactive end groups. The substrate diffusion region forms open channels for mass transfer of triacylglycerol, diacylglycerol, monoacylglycerol, and glycerol.

2. The immobilized lipase according to claim 1, characterized in that, The carbon core has a particle size of 150–300 μm, and the total thickness of the silica shell is 1–20 μm; the average pore sizes of the water activity buffer zone, the lipase immobilization zone, and the substrate diffusion zone are 2–6 nm, 10–25 nm, and 30–80 nm, respectively.

3. The immobilized lipase according to claim 1 or 2, characterized in that, The mass ratio of trehalose to sorbitol is 1:(0.8-1.2), and the composite buffer phase formed by the silica gel network, trehalose, and sorbitol accounts for 3-6% of the total mass of the immobilized lipase; the total water content of the immobilized lipase after being equilibrated at 25°C is 2.5-4.5% by mass, and the immobilized protein loading is 35-55 mg / g dry carrier.

4. The immobilized lipase according to any one of claims 1 to 3, characterized in that, The hydrophobic adsorption group is C4-C18 alkyl, phenyl, benzyl, alkylphenyl or a combination thereof, preferably C8 alkyl; the flexible organic spacer arm includes a polyether chain, and the reactive end group is epoxy, aldehyde, activated carboxyl, maleimide or a combination thereof, preferably epoxy.

5. A method for preparing the radially partitioned water activity buffered immobilized lipase according to any one of claims 1 to 4, characterized in that, include: S1, forming a porous silica layer with inner channels on the surface of a carbon core and removing the template agent of the inner channels; S2, the porous silica layer is impregnated with an impregnation solution containing trehalose, sorbitol and oligosilicic acid that can enter the inner pores, and the oligosilicic acid is polycondensed in the inner pores to form a silica gel network, thereby forming a water activity buffer. S3, a lipase immobilization region is formed on the outer side of the water activity buffer zone, and a hydrophobic adsorption group and a flexible organic spacer arm containing reactive end groups are introduced on the pore surface of the lipase immobilization region. S4, forming a substrate diffusion region with an average pore size larger than that of the lipase immobilization region on the outer side of the lipase immobilization region. S5, the lipase enters the lipase immobilization region through the substrate diffusion region, first adsorbs at the interface formed by the hydrophobic adsorption group, and then undergoes secondary immobilization with the reactive end group of the flexible organic spacer arm. S6, the obtained immobilized lipase is subjected to humidity balancing.

6. The preparation method according to claim 5, characterized in that, The oligomeric silicate in step S2 is formed by pre-hydrolysis of tetraethyl orthosilicate or by acidification of soluble silicate; the volume of the single impregnation liquid is 70-95% of the pore volume of the inner porous silica layer obtained in step S1, and 2-3 impregnations are performed; after each impregnation, gelation is performed at 35-45°C, and aging and drying are performed before the next impregnation or step S3.

7. The preparation method according to claim 5 or 6, characterized in that, Step S3 first forms a mesoporous silica layer with an average pore size of 10-25 nm outside the water activity buffer zone, and introduces C8 alkyl hydrophobic groups and polyether flexible spacer arms with epoxy end groups on the pore surface of the mesoporous silica layer through co-condensation, post-grafting or a combination thereof; Step S4 forms an open macroporous silica layer with an average pore size of 30-80 nm outside the mesoporous silica layer.

8. The preparation method according to any one of claims 5 to 7, characterized in that, The template agent used in steps S3 and S4 to form the silica layer is removed by solvent extraction.

9. A method for preparing diacylglycerol oil using the immobilized lipase according to any one of claims 1 to 4, characterized in that, The vegetable oil and glycerol were subjected to glycerolysis under the catalysis of the immobilized lipase. The molar ratio of triacylglycerol to glycerol was 1:(0.50-0.70) based on the triacylglycerol in the vegetable oil. The reaction temperature was 55-65℃. Glycerol was added in 2-4 portions or continuously over 4-6 hours.

10. The method according to claim 9, characterized in that, The immobilized lipase was packed into a fixed-bed reactor at a reaction temperature of 58–62°C and an empty bed residence time of 1.0–2.5 h. Pre-dehydrated vegetable oil and glycerol were continuously or intermittently supplied. Before the reaction, the immobilized lipase was pre-equilibrated at 25°C to a water activity (aw) of 0.35–0.42 and a total water content of 2.5–4.5% by mass.

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