A kind of immobilized lipase based on composite modified silica and its ugi multi-component reaction preparation method and application

Abstract

The application belongs to the technical field of immobilized enzyme, and discloses an immobilized lipase based on composite modified silica, a preparation method and application thereof. The immobilized lipase takes mesoporous silica as a carrier, and the surface of the carrier is simultaneously modified with octadecyl hydrophobic groups and free aldehyde groups. The lipase is covalently condensed with the aldehyde groups and isonitrile cross-linking agents through Ugi four-component reaction to form an irreversible amide covalent skeleton and is immobilized on the surface of the carrier. The application further provides a preparation method of the immobilized lipase and application thereof in catalyzing glycerolysis reaction to produce mono-diglycerol fatty acid esters. The application has the advantages of high enzyme activity recovery rate, good reusability and the like, and is suitable for the field of oil and fat deep processing.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, specifically relating to an immobilized lipase based on composite modified silica and its Ugi multi-component reaction preparation method and application. Background Technology

[0002] Improving dietary fat structure to prevent obesity and cardiovascular disease has become a consensus. Triglycerides (TAG), the main component of conventional edible oils, are easily stored as body fat; however, the intake of diglycerides (DAG) can significantly inhibit fat accumulation. When combined with an appropriate amount of monoglycerides (MAG), a complex lipid—namely, mono- and diglyceride fatty acid esters—can produce a significant synergistic effect. In this system, the dominant DAG provides the core health-promoting fat-reducing effect; while an appropriate amount of MAG acts as a powerful endogenous co-emulsifier and crystallization modifier, greatly improving the "self-emulsification" stability and processing texture of oils in complex water-oil systems. These mono- and diglyceride fatty acid esters, possessing both healthy physiological functions and excellent physicochemical properties, have extremely broad application prospects in high-end healthy edible oils, zero-trans-fatty acid plastic fats (such as shortening and margarine), functional food compound emulsifiers, and pharmaceutical and daily chemical delivery carriers. Therefore, converting traditional TAG into high-value-added mono- and diglyceride fatty acid esters and developing multifunctional healthy structural lipids has a promising development trend. The main industrial pathway for converting TAG into mono- and diglyceride fatty acid esters is glycerolysis. Compared to traditional chemical methods that are energy-intensive and prone to producing byproducts, enzymatic glycerolysis is mild, environmentally friendly, and pollution-free. However, free lipases are easily denatured and inactivated in the oil-glycerol two-phase system and are difficult to recycle. Immobilizing them on high-performance carriers is key to overcoming industrialization bottlenecks.

[0003] Mesoporous silica (such as SBA-15) is an ideal immobilization carrier due to its large specific surface area and ease of modification. However, existing technologies for immobilizing lipases for DAG production still have significant drawbacks: First, lipases require extremely strong hydrophobic interfaces to induce "interfacial activation," and conventional carriers suffer from low catalytic efficiency due to insufficient hydrophobic microenvironment. Second, existing covalent cross-linking strategies, such as Schiff bases or epoxy groups, are prone to reversible hydrolysis in glycerol hydrolysis systems, leading to chronic detachment of enzyme molecules and a short operating half-life. Third, conventional oxidation and high-temperature drying processes during the preparation of strongly hydrophobic microporous carriers easily cause "capillary condensation lock-up" of hydrophobic channels, resulting in thermal deactivation of highly active free aldehyde groups. Therefore, overcoming the defects of easy hydrolysis of chemical bonds in existing immobilized carriers and constructing immobilized lipases with an extremely stable covalent backbone while maintaining a strongly hydrophobic microenvironment through innovative carrier preparation processes is a key technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the problems of insufficient stability, low loading, low enzyme activity, low enzyme recovery rate and easy hydrolysis of immobilized enzymes in the prior art. The present invention provides a method for using an immobilized lipase with octadecyl and aldehyde groups modified silica as a carrier and using it in the catalytic reaction of TAG glycerolysis to produce mono- and diglyceride fatty acid esters.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for preparing immobilized lipase based on composite-modified silica includes the following steps: (1) Mesoporous silica, hydrophobic modifier, covalent modifier and organic base are subjected to silanization reaction in the first organic solvent, and the precursor carrier is obtained by solid-liquid separation, first washing and drying; (2) The precursor carrier obtained in step (1) is placed in an acidic solvent for ring-opening reaction. After solid-liquid separation and second washing, sodium periodate solution and tert-butanol are added for oxidation reaction. After the reaction is completed, the carrier is washed in the third time and freeze-dried to obtain the composite modified silica carrier. (3) The composite modified silica carrier obtained in step (2) is dispersed in the lipase solution for interfacial adsorption, and then an isonitrile crosslinking agent is added for Ugi reaction. After solid-liquid separation and fourth washing, the immobilized lipase is obtained.

[0007] In some embodiments, in step (1), the mesoporous silica is any one of SBA-15 type mesoporous silica, MCM-41 type mesoporous silica, or MCM-48 type mesoporous silica, preferably SBA-15 type mesoporous silica; and / or, the hydrophobic modifier is any one or more combinations of 3-(trimethoxysilyl)methacrylate, N-octadecyltriethoxysilane, triethoxy(octyl)silane, triethoxymethylsilane, and (3-bromopropyl)trimethoxysilane, preferably 3-(trimethoxysilyl)methacrylate, N-octadecyltriethoxysilane, or triethoxy(octyl)silane. The covalent modifier is any one of alkylene, triethoxymethylsilane, and (3-bromopropyl)trimethoxysilane, more preferably any one of 3-(trimethoxysilyl)propyl methacrylate, N-octadecyltriethoxysilane, triethoxy(octyl)silane, and triethoxymethylsilane, most preferably N-octadecyltriethoxysilane; and / or, the covalent modifier is 3-glycidoxypropyltrimethoxysilane; and / or, the organic base is any one of triethylamine, pyridine, or N,N-diisopropylethylamine, preferably triethylamine; and / or, the first organic solvent is any one of toluene, cyclohexane, isopropanol, or xylene, preferably toluene.

[0008] The hydrophobic modifiers used in this invention—3-(trimethoxysilyl)methacrylate, N-octadecyltriethoxysilane, triethoxy(octyl)silane, triethoxymethylsilane, and (3-bromopropyl)trimethoxysilane—can respectively esterify, octadecylate, octylate, methylate, and bromopropylate silicon dioxide; the covalent modifier 3-glycidoxypropyltrimethoxysilane can epoxylate silicon dioxide, followed by aldehyde modification based on the epoxy groups.

[0009] In some embodiments, in step (1), the sum of the amounts of the hydrophobic modifier and the covalent modifier to the mesoporous silica has a mass ratio of 1-5 mmol:1g, preferably 3 mmol:1g; and / or, the molar ratio of the hydrophobic modifier to the covalent modifier is 1-4:1-4, preferably 1:1-4, and most preferably 1:1; and / or, the mass-to-volume ratio of the mesoporous silica to the organic base is 1g:100-300 μL, preferably 1g:150 μL; and / or, the mass-to-volume ratio of the mesoporous silica to the first organic solvent is 1g:10-60 mL, preferably 1g:30 mL; and / or, the silanization reaction is carried out in a nitrogen atmosphere at 70-105°C and 200-250°C. The reaction is carried out at rpm for 4-8 h; and / or the first washing is performed using tetrahydrofuran; and / or the drying is performed at a temperature of 80-120 °C for 4-10 h.

[0010] In some embodiments, in step (2), the acidic solvent is a sulfuric acid solution with a concentration of 0.05~0.2 mol / L, preferably 0.1 mol / L; and / or, the mass-to-volume ratio of the precursor carrier to the acidic solvent is 1 g: 40~50 mL, preferably 1 g: 30 mL; and / or, the ring-opening reaction is carried out at a temperature of 40℃~60℃ for 5h~7h and at a rotation speed of 150rpm~300rpm; and / or, the second washing is performed using water; and / or, the concentration of the sodium periodate solution is 0.1~0.3 mol / L, preferably 0.1 mol / L; and / or, the mass-to-volume ratio of the precursor carrier to the sodium periodate solution is 1 g: 15~25 mL, preferably 1 g: 17.5 mL; and / or, the mass-to-volume ratio of the precursor carrier to the tert-butanol is 1 g: 4~6 mL, preferably 1 g: 5 mL. mL; and / or, the oxidation reaction is carried out under light-protected conditions at 20~25℃ and 200~300 rpm for 4~6 h with stirring; and / or, the third washing is washing with water and ethanol; and / or, the freeze drying is carried out at a temperature of -110~-80℃ for 10~12 h and a pressure of 20~30 Pa.

[0011] In some embodiments, in step (2), the oxidation reaction process is monitored by potassium iodide titration to determine the concentration change of sodium periodate in the system and thus the reaction endpoint is determined; preferably, the freeze-drying is carried out at a temperature of -110°C for 12 hours and a pressure of 20 Pa; the freeze-drying removes the moisture inside the pores under vacuum sublimation at extremely low temperatures to prevent the aldehyde group from undergoing aldol condensation or excessive oxidation under thermal drive, thereby maintaining 100% monomeric reactivity of the free aldehyde group.

[0012] In some embodiments, in step (3), the lipase is Thermomyces lanuginosus lipase (TLL), Candida Antarcticalipase B (CALB), Rhizomucor miehei lipase (RML), or Porcine pancreatic lipase (PPL), preferably Thermomyces lanuginosus lipase; preferably, the specific enzyme activity of the lipase is 300~400 U / g, and 1 unit (U) of lipase activity is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of p-nitrophenyl palmitate (pNPP) to p-nitrophenol (pNP) per minute.

[0013] In some embodiments, in step (3), the isonitrile crosslinking agent is an alicyclic isonitrile or a branched alkyl isonitrile. Preferably, the isonitrile crosslinking agent is any one or more combinations of 2-morpholinoethyl isonitrile, 2-isocyanonaphthalene, cyclohexyl isonitrile, benzyl isonitrile, 1-isocyanosulfonane, 1,1,3,3-tetramethylbutyl isonitrile, ethyl isocyanate, or p-toluenesulfonylmethyl isonitrile, preferably cyclohexyl isonitrile; and / or, the concentration of the lipase solution is 0.04~0.5 g / L, preferably 0.15~0.5 g / L; and / or, the mass-to-volume ratio of the composite modified silica carrier to the lipase solution is 1~2.5 mg:1 mL; and / or, the mass-to-volume ratio of the composite modified silica carrier to the isonitrile crosslinking agent is 1~2.5 mg:1 mL. mg: 1 μL; and / or, the Ugi reaction is carried out at 35~45℃ and 150~200rpm for 3~5h; the fourth wash is a wash with water.

[0014] The immobilized lipase based on composite modified silica prepared by the above method is also within the scope of protection of this invention.

[0015] This invention also protects the application of the above-mentioned immobilized lipase based on composite modified silica in the catalytic glycerolysis reaction of triglycerides to prepare mono- and diglyceride fatty acid esters; preferably, the mono- and diglyceride fatty acid esters are mono- and diglyceride oleate esters.

[0016] In some embodiments, the glycerolysis reaction includes: mixing triglycerides, glycerol, a second organic solvent, and the immobilized lipase, followed by centrifugation after the reaction to obtain mono- and diglyceride fatty acid esters.

[0017] In some embodiments, the second organic solvent is tert-butanol; and / or, the mass ratio of the immobilized lipase to the triglyceride is 0.1~1g:1g; and / or, the molar ratio of the triglyceride to the glycerol is 1:0.5~3, preferably 1:1; and / or, the amount of the second organic solvent added is 5%~30% of the mass of the triglyceride, preferably 10%; and / or, the reaction is carried out at 45~55°C and 200~300rpm for 2~24h.

[0018] This invention provides a composite-modified silica support, in which the surface of mesoporous silica is subjected to hydrophobic and covalent modifications, simultaneously achieving interfacial activation and covalent anchoring to improve the immobilization rate and activity of immobilized lipase. The hydrophobic modification is octadecylation, and the covalent modifier is aldehyde modification. The composite-modified silica support and isonitrile crosslinking agent are added together to a mixed aqueous solution containing lipase for stirring reaction. The hydrophobic regions on the lipase surface first undergo strong hydrophobic adsorption with the C18 long chain of the support, inducing the lipase to enter the "interfacial activated" open conformation. Subsequently, the aldehyde groups on the support surface and the isonitrile groups in the system undergo a highly specific Ugi four-component reaction (U-4CR) with the free amino and carboxyl groups on the lipase surface, constructing an irreversible amide bond skeleton and achieving extremely strong covalent anchoring. Therefore, the hydrophobic groups and aldehyde groups on the composite-modified silica support synergistically enhance the enzyme activity of lipase. The immobilized lipase prepared using it as a support not only has further enhanced enzyme activity but also better stability.

[0019] The composite-modified immobilized lipase provided by this invention can be efficiently applied to catalyze the glycerolization reaction of lipids (triglycerides) to prepare mono- and diglyceride fatty acid esters with healthy structures. Specifically, the process involves using lipids and glycerol as substrates, tert-butanol as the reaction medium, and conducting a transesterification reaction catalyzed by the immobilized lipase. The immobilized enzyme of this invention exhibits extremely high catalytic activity and operational stability at this complex interface of strong polarity (glycerol) and strong hydrophobicity (lipids), effectively suppressing hydrolysis side reactions and significantly improving the structural selectivity and final yield of mono- and diglyceride fatty acid esters, demonstrating great potential for industrial application.

[0020] Beneficial effects:

[0021] (1) The innovative mild preservation preparation process achieves high-density retention of highly active aldehyde groups. In the carrier preparation stage, the present invention uses a slightly acidic tert-butanol mixed water system for specific oxidation, combined with freeze-drying process, which completely solves the problems of "capillary condensation lock-up" of strong hydrophobic channels and thermal deactivation of free aldehyde groups in traditional processes, and perfectly maintains the permeability of the channel structure.

[0022] (2) The strong hydrophobic microenvironment significantly enhances the catalytic activity of the enzyme. The high-density octadecyl (C18 long chain) modified on the surface of the carrier provides an excellent hydrophobic interface for lipase, which can effectively induce the lipase "cap" to open and achieve "interface activation"; at the same time, it can repel polar byproducts in the reaction system and greatly promote the mass transfer of the substrate triglyceride.

[0023] (3) The Ugi multicomponent reaction endows the immobilized enzyme with extreme covalent stability. This invention abandons the traditional easily hydrolyzed Schiff base or epoxy cross-linking and innovatively introduces an isonitrile cross-linking agent to trigger the Ugi four-component reaction. This reaction constructs an irreversible amide bond in one step, firmly locking the activated lipase on the carrier and completely solving the problem of chronic enzyme molecule shedding in long-cycle glycerol hydrolysis reactions.

[0024] (4) It catalyzes the conversion of triglycerides with high efficiency, which greatly reduces the cost of industrial production. The prepared immobilized lipase exhibits extremely high yield and structural selectivity in the catalytic preparation of mono- and diglyceride fatty acid esters; and can be rapidly separated from high-viscosity oil phase systems by simple physical means (such as centrifugation or filtration), which has extremely high industrial application value.

[0025] (5) A highly versatile immobilization platform was constructed, expanding the broad applicability of the carrier. The highly active aldehyde-modified mesoporous carrier prepared by this invention not only perfectly matches the customized needs of lipase through its hydrophobic microenvironment, but its Ugi cross-linking mechanism is also universally applicable to any biomacromolecule with free amino and carboxyl groups on its surface (such as other industrial enzymes, antibodies, or large model protein molecules).

[0026] (6) The silica immobilized enzyme modified with aldehyde group and octadecyl group has a high loading capacity, up to 75 mg / g, and a specific enzyme activity up to 23482 U / g. Attached Figure Description

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0028] Figure 1Infrared spectra of the original SBA-15, 11-SBA-15 (aldehyde-octadecyl-SBA-15), and 11-SBA-15-TLL (aldehyde-octadecyl-SBA-15-TLL).

[0029] Figure 2 This is a statistical graph showing the relative activity and protein load of immobilized lipase TLL prepared using different molar ratios of covalent and hydrophobic modifiers in Example 2.

[0030] Figure 3 This is a statistical graph showing the specific activity and activity recovery rate of the immobilized lipase TLL prepared using different molar ratios of covalent and hydrophobic modifiers in Example 2.

[0031] Figure 4 This is a statistical chart showing the specific activity and protein load of the immobilized lipase TLL prepared using different cross-linking agents in Example 3.

[0032] Figure 5 This is a statistical graph showing the enzyme activity retention rate of the immobilized lipase TLL prepared in Example 4 at different temperatures.

[0033] Figure 6 The yield of mono- and diglyceride oleate produced by immobilized lipase TLL under different triglyceride to glycerol molar ratios in Example 5 is shown.

[0034] Figure 7 The yield of mono- and diglycerides produced by immobilized lipase TLL under different amounts of tert-butanol in Example 5 is shown.

[0035] Figure 8 The yields of mono- and diglyceride oleates produced using immobilized lipase TLL under different reaction time conditions in Example 5 are shown. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0037] In this embodiment of the invention, the experimental methods described are conventional methods unless otherwise specified; the reagents described are commercially available unless otherwise specified.

[0038] This invention utilizes hydrophobic modifiers such as N-octadecyltriethoxysilane and covalent modifiers containing epoxy groups such as 3-glycidoxypropyltrimethoxysilane to first modify silica with octadecyl groups and epoxy groups. Then, the epoxy groups are subjected to ring-opening oxidation treatment to obtain an octadecyl-aldehyde-silica support. Cyclohexylisocyanate is added, and lipase is immobilized on the surface of the composite-modified silica support through covalent bonding and hydrophobic adsorption.

[0039] This invention specifically selects aldehyde groups as anchoring points on the carrier surface. Only a mild isocyanate crosslinking agent needs to be introduced to spontaneously initiate the UGI reaction under green and mild conditions. Furthermore, the method is simple to operate, the carrier has a high lipase loading capacity, and the immobilized lipase has high enzyme activity and enzyme activity recovery rate, as well as good stability. It can be used in large-scale continuous operation for the production of DAG.

[0040] Example 1: Preparation of aldehyde / octadecyl composite modified silica support

[0041] (1) Grafting reaction: Weigh 1g of SBA-15 type mesoporous silica (purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., item number 103002) and add it to 30mL of anhydrous toluene. Then add 150μL of triethylamine, and then add a total of 3mmol of 3-glycidoxypropyltrimethoxysilane (covalent modifier) ​​and N-octadecyltriethoxysilane. The molar ratios of the two are 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:1, respectively. Stir the reaction at 95℃ and 200rpm for 4h under a nitrogen atmosphere. Centrifuge the reaction solution, wash the solid precipitate several times with tetrahydrofuran, and dry it under vacuum at 120℃ for 8h to obtain solid powder.

[0042] (2) Ring opening with dilute acid: 1g of the above solid powder was added to 30 mL of 0.1mol / L dilute sulfuric acid solution and stirred in a shaker at 50℃ and 200rpm for 6h. The reaction solution was centrifuged and washed several times with pure water with a pH of 7 to obtain a moist vicinal diolized support.

[0043] (3) Oxidation of sodium periodate: The above-mentioned vicinal diolized carrier was suspended in 17.5 mL of 0.1 mol / L NaIO4 solution, 5 mL of tert-butanol was added, and the mixture was stirred at 200 rpm for 4 hours in a shaker at room temperature in the dark.

[0044] (4) Reaction treatment: Samples were taken every 1 h, and after adding a weakly alkaline buffer, titration was performed using a starch-potassium iodide solution combined with sodium thiosulfate. The oxidation endpoint was determined by calculating the amount of sodium periodate consumed. After the reaction was completed, the sample was washed alternately with deionized water at pH 7 and ethanol until no unreacted periodate ions were found. The obtained solid was freeze-dried at -110℃ and 20Pa for 12 h to obtain a highly active composite modified support. The composite modified silica support prepared when the addition ratio of 3-glycidoxypropyltrimethoxysilane (epoxy covalent modifier) ​​and N-octadecyltriethoxysilane (hydrophobic modifier) ​​was 1:1 was named 11-SBA-15.

[0045] Example 2: Preparation of immobilized lipase by Ugi reaction

[0046] 20 mg of each of the seven carriers prepared in Example 1 was weighed and added to 9 mL of water. After uniform dispersion, 1 mL of lipase (TLL lipase) containing 5 mg / mL protein was added to the system, followed by 10 μL of cyclohexylisocyanate as a cross-linking agent. The mixture was stirred at 40 °C and 200 rpm for 4 hours to ensure sufficient contact between the aldehyde, isonitrile, amino, and carboxyl groups and to complete U-4CR condensation. The solid was separated, and the supernatant was retained. The solid was washed several times with 10 mL of pure water, and then resuspended in 10 mL of pure water to obtain the suspensions of the seven immobilized lipases.

[0047] Material characterization and performance testing

[0048] 1. Elemental analysis test

[0049] Elemental analysis was performed on the original SBA-15 mesoporous silica, the 11-SBA-15 prepared in Example 1, and the immobilized lipase TLL prepared using 11-SBA-15 as a carrier in this example. The results are shown in Table 1. Compared with the original SBA-15 mesoporous silica, the C and H content of the composite modified silica (11-SBA-15) increased significantly, indicating that the octadecyl hydrophobic modifier and the aldehyde covalent modifier were successfully grafted onto the silica. Compared with the composite modified silica (11-SBA-15), the N content of the immobilized enzyme increased significantly, indicating that the lipase was successfully immobilized on the carrier.

[0050] Table 1. Elemental analysis of original SBA-15 mesoporous silica, 11-SBA-15, and immobilized lipase TLL.

[0051]

[0052] 2. Infrared spectroscopy analysis

[0053] like Figure 1As shown, the original SBA-15, 11-SBA-15, and 11-SBA-15-TLL at 1080 cm⁻¹ -1 and 810 cm -1 The presence of extremely strong Si-O-Si characteristic vibrational peaks at all locations demonstrates that the basic silica mesoporous framework of SBA-15 was well preserved after modification and immobilization of lipase. Compared with the original SBA-15, the composite-modified carrier exhibited significantly higher vibrational peaks at 2926 cm⁻¹. -1 and 2866 cm -1 A distinct -CH2- vibrational peak was added at 1655 cm⁻¹. -1 The characteristic -C=O absorption peak appeared at [location missing], and these two significant changes conclusively prove that the octadecyl hydrophobic long carbon chain and aldehyde group have been successfully grafted onto the support surface. Furthermore, the infrared spectrum after immobilization of the lipase shows that the system spectrum [missing information] at 1536 cm⁻¹. -1 The presence of a specific -CN (amide II band) absorption peak, representing a protein peptide bond, provides molecular evidence for the successful covalent binding and immobilization of lipase TLL on the composite modified vector.

[0054] 3. Colorimetric test of 2,4-dinitrophenylhydrazine

[0055] 20 mg of the original SBA-15 mesoporous silica and 11-SBA-15 prepared in Example 1 were weighed and added to 2,4-dinitrophenylhydrazine (2,4-DNPH). When the pure white composite modified support (11-SBA-15) powder was immersed in the colorless 2,4-DNPH reagent, a significant color reaction rapidly occurred on the powder surface, exhibiting a strong bright yellow color, while the unmodified original SBA-15 control group showed no color change. This demonstrates the successful modification of the aldehyde group on the SBA-15 support.

[0056] 4. Protein loading, specific enzyme activity, relative activity, and enzyme activity recovery rate tests

[0057] (1) Determine the protein loading of the above 7 immobilized lipases TLL: The protein content P1 in the supernatant obtained after centrifugation of the above reaction solution was determined by Coomassie brilliant blue method, and the protein loading of immobilized lipase TLL was calculated by the following formula.

[0058]

[0059] Where P0 is the amount of lipase TLL used in the preparation of immobilized lipase TLL (mg), P1 is the mass of lipase TLL in the supernatant (mg), and S is the amount of composite modified silica used in the preparation of immobilized lipase TLL (g).

[0060] (2) The specific activity, relative activity, and activity recovery rate of the above seven immobilized lipases TLL were determined using the following methods:

[0061] Take 1.9 mL of p-nitrophenyl palmitate (pNPP) solution, preheat at 50 °C and 200 rpm for 2 min, then add 0.1 mL of the suspensions of the above seven immobilized lipase TLLs and the original lipase TLL solution used in the preparation process. After reacting for 20 min, measure the absorbance of the reaction solution at a wavelength of 410 nm, and calculate the enzyme activity (U / L) of the seven immobilized lipase TLLs using a standard curve. I ) and free lipase TLL enzyme activity (U L Lipase activity unit (U) is defined as the amount of lipase required to catalyze the conversion of 1 μmol pNPP to p-nitrophenol (pNP) per minute. The specific activity, relative activity, and activity recovery of the immobilized lipase TLL are calculated using the following formulas:

[0062]

[0063] Among them, U I The enzyme activity of 0.1 mL of the above immobilized lipase TLL suspension is expressed in units of U / M. I The mass of lipase loaded in 0.1 mL of the above immobilized lipase TLL suspension is expressed in g; U L The activity of the above-mentioned free lipase TLL in 0.1 mL is expressed in units of U; M. L The mass of 0.1 mL of the above free lipase TLL is expressed in g.

[0064] The pNPP solution preparation method is as follows: Weigh 0.0755g of pNPP and dilute to 10mL with isopropanol (dissolved in a 50℃ water bath). Add a small amount of PBS buffer (pH=7.0, 50mM) to a 500mL volumetric flask. Pour the isopropanol solution of pNPP into the volumetric flask, add 2g of Tritiumx-100, and sonicate at 100W for 15min until it turns milky white. Dilute to 500mL with PBS and pour into a brown bottle. Continue sonicating at 100W for 15min, and then store in a 4℃ refrigerator protected from light for later use.

[0065] The results of the determination of specific enzyme activity, relative enzyme activity, enzyme activity recovery rate, and protein loading of the above seven immobilized lipases TLL are as follows: Figure 2 , Figure 3As shown, the results indicate that the specific enzyme activity of immobilized lipase TLL is increased to varying degrees compared with that of free lipase TLL. With changes in the ratio of covalent modifier to hydrophobic modifier, the protein loading, lipase activity recovery rate, relative activity, and specific enzyme activity of immobilized lipase TLL all changed to varying degrees. When the molar ratio of covalent modifier (aldehyde group) to hydrophobic modifier (octadecyl group) was 1:4, the immobilized enzyme TLL exhibited the highest protein loading of 69 mg / g; when the molar ratio of covalent modifier to hydrophobic modifier was 1:1, the immobilized enzyme TLL exhibited the highest relative enzyme activity (4394%), and the protein loading was also relatively high at 51 mg / g. Furthermore, compared with the carrier that only undergoes hydrophobic modification and the immobilized lipase that only undergoes covalent modification, the specific enzyme activity and enzyme activity recovery rate of the immobilized enzyme were significantly improved when the molar ratio of covalent modifier to hydrophobic modifier was 1:1, indicating that the covalent modifier and hydrophobic modifier synergistically enhanced the enzyme activity of the immobilized enzyme.

[0066] Example 3: Optimization of crosslinking agent

[0067] Weigh 20 mg of the composite modified silica 11-SBA-15 prepared in Example 1 and add it to 10 mL of lipase TLL solution with a protein concentration of 0.15 mg / mL. Then add 10 μL of 2-isocyanonaphthalene, cyclohexylisocyanate, 1-isocyanoacetane, 4-bromophenylisocyanate, 1,1,3,3-tetramethylbutylisocyanate, ethyl isocyanate and p-toluenesulfonylmethylisocyanate respectively. Stir the mixture at 40 °C and 200 rpm for 4 h. After the reaction is completed, centrifuge the reaction solution, retain the supernatant, wash the solid precipitate several times with pure water, and resuspend it in 10 mL of pure water to obtain a suspension of 7 immobilized lipase TLLs.

[0068] The relative activity of the seven immobilized lipases TLL prepared in this example was determined according to the method in Example 2.

[0069] Experimental results are as follows Figure 4As shown, with the addition of isonitrile crosslinking agents, a highly specific Ugi four-component reaction (U-4CR) can occur. The aldehyde groups on the carrier surface, the isonitrile groups in the system, and the free amino and carboxyl groups on the lipase surface undergo multi-component condensation simultaneously to construct an amide covalent backbone. Based on the construction of this 'absolutely locked' active conformation, isonitrile compounds can form a strong covalent network as crosslinking agents. Because ethyl isocyanate and p-toluenesulfonylmethyl isonitrile have highly polar ester and sulfonyl groups that disrupt the hydrophobic environment, these two crosslinking agents are less effective at enhancing lipase activity than other isocyanate crosslinking agents with hydrophobic groups such as cyclic side chains. However, most of the crosslinking agents with hydrophobic side chains used in this example have excellent enzyme activity enhancement effects, among which cyclohexyl isonitrile has the best effect, increasing the enzyme activity to about 23482 U / g, and the protein loading can also reach about 70 mg / g.

[0070] Example 4: Determination of temperature stability

[0071] 20 mg of each of the following were weighed separately: the composite modified silica 11-SBA-15 prepared in Example 1 with a molar ratio of covalent modifier (3-glycidoxypropyltrimethoxysilane) and hydrophobic modifier (N-octadecyltriethoxysilane) of 1:1, and the silica carrier with bifunctional epoxy and octadecyl modification without dilute acid ring-opening and oxidation modification. They were added to 10 mL of lipase TLL solution with a protein concentration of 0.15 mg / mL. Then, 10 μL of cyclohexylisocyanate was added to each. The mixture was stirred at 40 °C and 200 rpm for 4 h. After the reaction was completed, the reaction solution was centrifuged, and the supernatant was retained. The solid precipitate was washed several times with pure water and then resuspended in 10 mL of pure water to obtain suspensions of the two immobilized lipase TLLs.

[0072] Determination of the temperature stability of immobilized lipase TLL and free TLL: The prepared 11-SBA-15-TLL-1, the enzyme immobilized on the bifunctional modified vector, and the free TLL lipase were incubated in water baths at 40, 50, 60, 70, 80, and 90°C for 2 hours, respectively. Then, the specific activity of the immobilized lipase TLL and the free enzyme was measured before incubation and after incubation at each temperature according to the method in Example 2. The enzyme activity retention rate of the two immobilized lipase TLLs under different incubation temperature conditions was then calculated.

[0073]

[0074] The results are as follows Figure 5As shown, under the Ugi multi-component reaction, the irreversible amide covalent backbone constructed between the support and the free amino and carboxyl groups on the lipase surface, 11-SBA-15-TLL-1, exhibits superior catalytic activity across all temperature gradients compared to both the bifunctional immobilized enzyme and the free enzyme. Particularly noteworthy is that under high-temperature heat stress of 70°C, the free enzyme completely denatures and becomes inactive, while the composite-modified immobilized enzyme prepared in this invention retains 58% of its catalytic activity and still retains 5% of its activity at 90°C, demonstrating excellent resistance to heat denaturation.

[0075] Example 5: Optimization of production conditions for triglyceride glycerol hydrolysis:

[0076] Raw materials: Triglycerides (chemically pure, 98.0~103.0, CAS No. 122-32-7, molecular weight 885.45, purchased from Sinopharm Chemical Reagent Co., Ltd.); Glycerol: analytical grade, purity ≥99%, molecular weight 92.1.

[0077] Weigh 0.5 g of the composite modified silica 11-SBA-15 prepared in Example 1, add it to 200 mL of lipase TLL solution with a protein concentration of 0.19 mg / mL, then add 250 μL of cyclohexylisocyanate, stir at 40 °C and 200 rpm for 1 h. After the reaction is completed, centrifuge the reaction solution, wash the solid precipitate several times with pure water, and freeze dry at -110 °C for 12 h to obtain 11-SBA-15-TLL-2. Weigh 1 g of the precipitate for triglyceride glycerolysis.

[0078] (1) Optimization of the ratio of triglycerides to glycerol

[0079] 1 g of triglyceride, 0.1 g of 11-SBA-15-TLL-2 prepared in this example, and 0.1 g of tert-butanol were added to a reactor. Glycerol was then added in molar ratios of triglyceride:glycerol of 1:0.5, 1, 1.5, 2.0, 2.5, and 3.0, respectively. The reaction was carried out at 45°C and 200 rpm for 24 h. After the reaction was completed, the reaction solution was centrifuged, the products were separated, and the yields of mono- and diglyceride oleates were calculated by high performance liquid chromatography.

[0080]

[0081] Among them, R 前 and R 后 These represent the peak areas of the triglyceride component in the HPLC chromatograms before and after the reaction.

[0082] The results are as follows Figure 6As shown, when the ratio of triglycerides to glycerol is 2, the TAG conversion rate increases less. Therefore, in order to balance the yield of mono- and diglyceride oleates and the cost of raw materials and maximize benefits, the optimal molar ratio of triglycerides to glycerol is 1, at which point the yield of mono- and diglyceride oleates is 81.6%.

[0083] (2) Optimization of tert-butanol dosage

[0084] 1 g of triglyceride, 0.1 g of 11-SBA-15-TLL-2 (prepared in this example), and 0.1 g of glycerol were weighed and added to a reactor. Tert-butanol was then added at 5%, 10%, 15%, 20%, and 30% of the triglyceride mass percentage, respectively. The reaction was carried out at 45°C and 200 rpm for 24 h with stirring. After the reaction, the reaction mixture was centrifuged, and the products were separated. The yields of mono- and diglyceride oleates were calculated using the area normalization method after high-performance liquid chromatography (HPLC). The results are as follows: Figure 7 As shown, the optimal mass percentage of tert-butanol is 10% of the triglycerides.

[0085] (3) Optimization of reaction time

[0086] 1 g of triglyceride, 0.1 g of 11-SBA-15-TLL-2 (prepared in this example), 0.1 g of glycerol, and 0.1 g of tert-butanol were weighed and added to a reactor. The reaction was carried out at 45°C and 200 rpm for 2, 4, 6, 8, 10, 12, and 24 h with stirring. After the reaction was completed, the reaction solution was centrifuged, the products were separated, and the yields of mono- and diglyceride oleates were calculated using the area normalization method after high-performance liquid chromatography. Figure 8 As shown, the yields of mono- and diglycerides gradually increased over time, reaching 85.75% at 20 h.

[0087] This invention provides a method for preparing and applying an immobilized lipase based on composite-modified silica and its Ugi multi-component reaction. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing immobilized lipase based on composite-modified silica, characterized in that, Includes the following steps: (1) Mesoporous silica, hydrophobic modifier, covalent modifier and organic base are subjected to silanization reaction in the first organic solvent, and the precursor carrier is obtained by solid-liquid separation, first washing and drying; (2) The precursor carrier obtained in step (1) is placed in an acidic solvent for ring-opening reaction. After solid-liquid separation and second washing, sodium periodate solution and tert-butanol are added for oxidation reaction. After the reaction is completed, the carrier is washed in the third time and freeze-dried to obtain the composite modified silica carrier. (3) The composite modified silica carrier obtained in step (2) is dispersed in the lipase solution for interfacial adsorption, and then an isonitrile crosslinking agent is added for Ugi reaction. After solid-liquid separation and fourth washing, the immobilized lipase is obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the mesoporous silica is any one of SBA-15 type mesoporous silica, MCM-41 type mesoporous silica, or MCM-48 type mesoporous silica; and / or, the hydrophobic modifier is any one or more combinations of 3-(trimethoxysilyl)methacrylate, N-octadecyltriethoxysilane, triethoxy(octyl)silane, triethoxymethylsilane, and (3-bromopropyl)trimethoxysilane; and / or, the covalent modifier is 3-glycidoxypropyltrimethoxysilane; and / or, the organic base is any one of triethylamine, pyridine, or N,N-diisopropylethylamine; and / or, the first organic solvent is any one of toluene, cyclohexane, isopropanol, or xylene.

3. The preparation method according to claim 1, characterized in that, In step (1), the sum of the amounts of the hydrophobic modifier and the covalent modifier is in a mass ratio of 1-5 mmol to 1g of the mesoporous silica; and / or, the molar ratio of the hydrophobic modifier to the covalent modifier is 1-4:1-4; and / or, the mass-volume ratio of the mesoporous silica to the organic base is 1g:100-300 μL; and / or, the mass-volume ratio of the mesoporous silica to the first organic solvent is 1g:10-60 mL; and / or, the silanization reaction is carried out in a nitrogen atmosphere at 70-105°C and 200-250 rpm for 4-8 h; and / or, the first washing is performed using tetrahydrofuran; and / or, the drying is performed at 80-120°C for 4-10 h.

4. The preparation method according to claim 1, characterized in that, In step (2), the acidic solvent is a sulfuric acid solution with a concentration of 0.05~0.2 mol / L; and / or, the mass-to-volume ratio of the precursor carrier to the acidic solvent is 1 g: 40~50 mL; and / or, the ring-opening reaction is carried out at a temperature of 40℃~60℃ for 5h~7h and a rotation speed of 150rpm~300rpm; and / or, the second washing is washing with water; and / or, the concentration of the sodium periodate solution is 0.1~0.3 mol / L; and / or, the mass-to-volume ratio of the precursor carrier to the sodium periodate solution is 1 g: 15~25 mL; and / or, the mass-to-volume ratio of the precursor carrier to the tert-butanol is 1 g: 4~6 mL; and / or, the oxidation reaction is carried out under light-protected conditions at 20~25℃ and 200~300 rpm. The reaction is stirred at rpm for 4-6 hours; and / or the third washing is performed using water and ethanol; and / or the freeze-drying is performed at a temperature of -110 to -80°C for 10-12 hours and a pressure of 20-30 Pa.

5. The preparation method according to claim 1, characterized in that, In step (3), the lipase is *Thermophilus spp.* lipase, *Candida antarcticus* lipase B, *Rhizopus miltiorrhiza* lipase, or porcine pancreatic lipase; and / or, the isonitrile crosslinking agent is an alicyclic isonitrile or a branched alkyl isonitrile, preferably, the isonitrile crosslinking agent is any one or more combinations of 2-morpholinoethyl isonitrile, 2-isocyanonaphthalene, cyclohexyl isonitrile, benzyl isonitrile, 1-isocyanate methyl ether, 1,1,3,3-tetramethylbutyl isonitrile, ethyl isocyanate, or p-toluenesulfonylmethyl isonitrile; and / or, the concentration of the lipase solution is 0.04~0.5 g / L; and / or, the mass-to-volume ratio of the composite modified silica carrier to the lipase solution is 1~2.5 mg:1 mL; and / or, the mass-to-volume ratio of the composite modified silica carrier to the isonitrile crosslinking agent is 1~2.5 mg:1 mL. mg: 1 μL; and / or, the Ugi reaction is carried out at 35-45°C and 150-200 rpm for 3-5 h; and / or, the fourth wash is a wash with water.

6. The immobilized lipase based on composite modified silica prepared by any one of the preparation methods of claims 1 to 5.

7. The application of the immobilized lipase based on composite modified silica as described in claim 6 in the catalytic glycerolysis of triglycerides to prepare mono- and diglyceride fatty acid esters.

8. The application according to claim 7, characterized in that, The mono- and diglyceride fatty acid esters are mono- and diglyceride oleate esters.

9. The application according to claim 7, characterized in that, The glycerol hydrolysis reaction includes: mixing triglycerides, glycerol, a second organic solvent, and the immobilized lipase, followed by centrifugation after the reaction to obtain mono- and diglyceride fatty acid esters.

10. The application according to claim 9, characterized in that, The second organic solvent is tert-butanol; and / or, the mass ratio of the immobilized lipase to the triglyceride is 0.1~1g:1g; and / or, the molar ratio of the triglyceride to the glycerol is 1:0.5~3; and / or, the amount of the second organic solvent added is 5%~30% of the mass of the triglyceride; and / or, the reaction is carried out at 45~55℃ and 200~300rpm for 2~24h.

Images