A method for encapsulating enzymes in situ with lignin

Abstract

The application discloses a method for in-situ encapsulating enzyme by lignin. The pH of industrial lignin water dispersion is adjusted to above 10.0 to dissolve the industrial lignin, and the industrial lignin solution is obtained after filtering and removing impurities. Then the pH of the industrial lignin solution is adjusted to 5-9.5, and enzyme protein is added to obtain a mixed solution. The pH of the mixed solution is adjusted to 3.0-4.0 again, so that the industrial lignin and the enzyme protein are co-precipitated. After separating the precipitate, drying is performed to obtain the immobilized enzyme. The application uses the adsorption capacity of lignin to enzyme and the property of alkali-soluble acid-extractable of lignin to immobilize the enzyme with low pH sensitivity, so that the encapsulation rate of the enzyme is improved, and the activity of the immobilized enzyme is better preserved. The application provides a new method for in-situ encapsulating enzyme protein, and has a positive significance for promoting the development of lignocellulose bio-refining and green bio-manufacturing.

Description

Technical Field

[0001] This invention relates to the technical field of immobilized enzymes, and more specifically, to a method for in-situ encapsulating enzymes using lignin. Background Technology

[0002] With the continuous development of biotechnology, enzymes, as highly efficient biocatalysts, have been widely used in various fields. However, enzymes often face problems such as poor stability, easy inactivation, and difficulty in recycling in practical applications. To solve these problems, researchers have been exploring various enzyme immobilization methods.

[0003] Currently, common enzyme immobilization methods include physical adsorption, covalent bonding, cross-linking, and encapsulation. Physical adsorption is simple to operate, but the binding force is weak, and the enzyme is prone to detachment. Covalent bonding results in a strong bond, but the reaction conditions are relatively harsh, which may affect enzyme activity. Cross-linking can improve enzyme stability, but it can also easily lead to a decrease in enzyme activity. Encapsulation can better maintain enzyme activity, but traditional encapsulation materials often have disadvantages such as high mass transfer resistance, low mechanical strength, and high cost.

[0004] Among existing enzyme immobilization methods, few can simultaneously meet the requirements of high stability, high activity, easy recyclability, and environmental friendliness. Furthermore, most existing encapsulation materials are synthetic materials, such as artificial polymers, metal-organic frameworks, and covalent organic frameworks, which have complex preparation processes, high costs, and may cause environmental pollution.

[0005] To address the challenges of enzyme immobilization, various methods have been proposed in existing technologies. Chinese invention patent CN1810867A discloses a method for encapsulating enzyme molecules using sodium alginate / chitosan hydrogel. While this method improves enzyme stability to some extent, the water-locking properties of the hydrogel can lead to significant mass transfer barriers between the enzyme and substrate, and the hydrogel's poor mechanical properties hinder its industrial application. Chinese invention patent CN117700696A discloses a method for encapsulating enzyme molecules using polylactic acid microspheres. Although this method significantly improves enzyme stability, the preparation of polylactic acid microspheres involves the use of organic solvents, which require prolonged evaporation for removal. This limits its applicability to different enzymes and hinders its widespread application.

[0006] In summary, there is an urgent need for a simple and efficient enzyme immobilization method that can overcome the shortcomings of existing methods, improve enzyme stability, activity and recyclability, and has the advantages of being environmentally friendly, simple to prepare and low in cost. Summary of the Invention

[0007] To overcome the aforementioned defects and shortcomings in the existing technology, the present invention provides a method for in-situ encapsulation of enzymes using lignin.

[0008] The first objective of this invention is to provide a method for in-situ encapsulation of enzymes using lignin.

[0009] A second objective of this invention is to provide an immobilized enzyme prepared by the method described above.

[0010] This invention claims protection for the following:

[0011] A method for in-situ encapsulating enzymes using lignin includes the following steps:

[0012] S1. Disperse industrial lignin in water to obtain a dispersion, adjust the pH of the dispersion to above 10.0, mix thoroughly and remove insoluble matter to obtain an industrial lignin solution;

[0013] S2. Adjust the pH of the industrial lignin solution to 5-9.5, then add enzyme protein, mix thoroughly to obtain a mixture;

[0014] S3. Adjust the pH of the mixture to 3.0-4.0 to co-precipitate industrial lignin and enzyme protein, then separate the precipitate and dry it to obtain the final product.

[0015] As one feasible approach, the industrial lignin is one or more of alkali lignin, enzymatically hydrolyzed lignin, organic solvent lignin, Klason lignin, and lignin extracted from plant materials using ionic liquids, deep eutectic solvents, or hydrated molten salt solvents.

[0016] Preferably, in step S1, the mass fraction of industrial lignin in the dispersion is 0.05-2%.

[0017] Preferably, in step S1, the thorough mixing is carried out at a temperature of 24–26°C.

[0018] As an feasible approach, in step S1, the pH is adjusted using an alkaline solution prepared from one or more of NaOH, KOH, Ca(OH)2, and ammonia.

[0019] Preferably, in step S2, the enzyme protein is one or more of glucose oxidase, β-glucosidase, alcohol dehydrogenase, glucose isomerase, glucose dehydrogenase, bromelain, lipase, catalase, protease, transaminase, glycosyltransferase, and phosphotransferase.

[0020] Preferably, in step S1, the mass fraction of industrial lignin in the dispersion is 0.1-2%; under this preferred scheme, the encapsulation rate of lignin-encapsulated enzymes in situ can be guaranteed to be ≥90%.

[0021] Preferably, in step S1, the pH of the dispersion is adjusted to 10.0–12.5; under this preferred scheme, the enzyme activity retention rate of the lignin-encapsulated enzyme can be guaranteed to be ≥75%.

[0022] More preferably, the industrial lignin is alkali lignin, the enzyme protein is bromelain, and the method includes the following steps:

[0023] S1. Disperse alkali lignin in water to obtain a dispersion, wherein the mass fraction of alkali lignin in the dispersion is 0.05-0.1%; adjust the pH value of the dispersion to above 10.0, mix thoroughly, and remove insoluble matter to obtain an alkali lignin solution;

[0024] S2. Adjust the pH of the alkali lignin solution to 7, then add bromelain and mix thoroughly to obtain a mixture;

[0025] S3. Adjust the pH of the mixture to 3.5 to co-precipitate alkali lignin and enzyme protein, then separate the precipitate and dry it to obtain the final product;

[0026] Under this preferred embodiment, the encapsulation rate of bromelain encapsulated using alkali lignin can reach 82-90%.

[0027] Most preferably, in step S1, the mass fraction of alkali lignin in the dispersion is 0.1%.

[0028] More preferably, the industrial lignin is alkali lignin, the enzyme protein is bromelain, and the method includes the following steps:

[0029] S1. Disperse alkali lignin in water to obtain a dispersion, wherein the mass fraction of alkali lignin in the dispersion is 0.05%; adjust the pH value of the dispersion to above 10.0, mix thoroughly, remove insoluble matter, and obtain an alkali lignin solution;

[0030] S2. Adjust the pH of the alkali lignin solution to 7, then add bromelain and mix thoroughly to obtain a mixture;

[0031] S3. Adjust the pH of the mixture to 3.5 to co-precipitate alkali lignin and enzyme protein, then separate the precipitate and dry it to obtain the final product;

[0032] Under this preferred embodiment, the loading rate of bromelain encapsulated with alkali lignin can reach 61-63%.

[0033] More preferably, the industrial lignin is alkali lignin, the enzyme protein is bromelain, and the method includes the following steps:

[0034] S1. Disperse alkali lignin in water to obtain a dispersion, wherein the mass fraction of alkali lignin in the dispersion is 0.05-0.1%; adjust the pH value of the dispersion to 10.0, mix thoroughly, and remove insoluble matter to obtain an alkali lignin solution;

[0035] S2. Adjust the pH of the alkali lignin solution to 5-7, then add bromelain and mix thoroughly to obtain a mixture;

[0036] S3. Adjust the pH of the mixture to 3.5-4 to co-precipitate alkali lignin and enzyme protein, then separate the precipitate and dry it to obtain the final product;

[0037] Under this preferred scheme, the enzyme activity retention rate of bromelain encapsulated with alkali lignin does not change significantly and can reach 82-85%.

[0038] As an implementable method, in steps S2 and S3, the pH is adjusted using an acid solution prepared from one or more of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and acetic acid.

[0039] Preferably, the mass of the enzyme protein is 20-200% of the mass of industrial lignin.

[0040] Preferably, the enzyme protein contains a protective agent.

[0041] Preferably, the protective agent is one or more of carboxymethyl cellulose, starch, maltodextrin, cyclodextrin, diatomaceous earth, glycerol, glucose, polyethylene glycol, polyvinylpyrrolidone, and polyvinyl alcohol.

[0042] Preferably, the mass of the protective agent is 20-400% of the enzyme protein mass.

[0043] Immobilized enzymes prepared by any of the methods described above.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention discloses a method for in-situ encapsulation of enzymes using lignin. This invention utilizes lignin's adsorption capacity for enzymes and its alkali-soluble and acid-precipitated properties to immobilize enzymes with low pH sensitivity. This not only improves the encapsulation rate but also better preserves the activity of the immobilized enzyme, achieving an enzyme activity retention rate of 72-104% after immobilization. Furthermore, this method is environmentally friendly, simple, efficient, and easy to promote and apply. This invention provides a novel method for in-situ encapsulation of enzyme proteins, which has positive significance for promoting the development of lignocellulose biorefining and green biomanufacturing. Attached Figure Description

[0046] Figure 1 This is a scanning electron microscope image of a lignin sample.

[0047] Figure 2 This is a scanning electron microscope image of an immobilized enzyme sample.

[0048] Figure 3 Scanning electron microscope image of nitrogen element distribution in immobilized enzyme sample. Detailed Implementation

[0049] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] Example 1: A method for in-situ encapsulation of enzymes based on lignin

[0052] The lignin used in this embodiment is enzymatically hydrolyzed lignin, which is derived from Shandong Longli Biotechnology Co., Ltd., and is extracted from the residue after preparing functional sugars from corn cobs.

[0053] The specific steps for using Longli enzymatic hydrolysis of lignin and in-situ encapsulation of enzymes are as follows:

[0054] S1. Add NaOH powder to 100g of an aqueous dispersion of 0.1% Longli enzymatic hydrolysed lignin, adjust the pH to 12.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0055] S2. Adjust the pH of the lignin solution to 7.0 with hydrochloric acid, then add 0.1g of glucose oxidase (derived from Aspergillus niger, with an enzyme activity of 100U / mg) containing 50% maltodextrin to the above system. Mix evenly at 24-26℃ to obtain a mixture.

[0056] S3. Adjust the pH of the mixture to 3.0 with hydrochloric acid to co-precipitate lignin and glucose oxidase. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then freeze-dry to obtain immobilized enzyme.

[0057] Example 2: A method for in-situ encapsulation of enzymes based on lignin

[0058] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0059] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0060] S1. Add KOH solid to 100g of an aqueous dispersion of 0.2% alkali lignin, adjust the pH to 12.5, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0061] S2. Adjust the pH of the lignin solution to 5.5 with sulfuric acid solution, then add 0.2g of liquid β-glucosidase (derived from almonds, enzyme activity 12U / mg) containing 70% glycerol by mass to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0062] S3. Adjust the pH of the mixture to 3.5 with sulfuric acid solution to co-precipitate lignin and β-glucosidase. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then freeze-dry to obtain the immobilized enzyme.

[0063] Example 3: A method for in-situ encapsulation of enzymes based on lignin

[0064] The lignin used in this embodiment is enzymatically hydrolyzed lignin, which is derived from Shandong Longli Biotechnology Co., Ltd., and is extracted from the residue after preparing functional sugars from corn cobs.

[0065] The specific steps for using Longli enzymatic hydrolysis of lignin and in-situ encapsulation of enzymes are as follows:

[0066] S1. Add KOH solid to 100g of 0.2% (w / w) Longli enzymatic hydrolysed lignin aqueous dispersion, adjust the pH to 12.5, let stand for 30min, filter to remove impurities, and obtain lignin solution;

[0067] S2. Adjust the pH of the lignin solution to 5.5 with sulfuric acid solution, then add 0.2g of liquid alcohol dehydrogenase (derived from Saccharomyces cerevisiae, enzyme activity 310U / mg) containing 80% glycerol by mass to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0068] S3. Adjust the pH of the mixture to 3.5 with sulfuric acid solution to co-precipitate lignin and alcohol dehydrogenase. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then freeze-dry to obtain immobilized enzyme.

[0069] Example 4: A method for in-situ encapsulation of enzymes based on lignin

[0070] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0071] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0072] S1. Add Ca(OH)2 solid to 100g of an aqueous dispersion of 1% alkali lignin, adjust the pH to 11.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0073] S2. Adjust the pH of the lignin solution to 5.5 with phosphoric acid solution, then add 1.0 g of glucose isomerase containing 50% starch (derived from Lactobacillus, enzyme activity 30 U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0074] S3. Adjust the pH of the mixture to 4.0 with phosphoric acid solution to co-precipitate lignin and glucose isomerase. Separate the precipitate by centrifugation at 8000 rpm for 10 min and vacuum drying at 40℃ to obtain immobilized enzyme.

[0075] Example 5: A method for in-situ encapsulation of enzymes based on lignin

[0076] The lignin used in this embodiment is ethanol lignin, which is extracted from corn stalks and obtained by extracting with a 60% ethanol aqueous solution at 160°C for 30 minutes.

[0077] The specific steps for in-situ encapsulation of enzymes using ethanol-lignin are as follows:

[0078] S1. Add ammonia to 100g of an aqueous dispersion of 2% ethanol lignin, adjust the pH to 12.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution.

[0079] S2. Adjust the pH of the lignin solution to 8.0 with sulfuric acid solution, and then add 5.0g of an aqueous solution containing 2% carboxymethyl cellulose and 10% glucose dehydrogenase (derived from Pseudomonas, enzyme activity 230U / mg) to the above system. Mix well at 24-26℃ to obtain a mixed solution.

[0080] S3. Adjust the pH of the mixture to 3.2 with sulfuric acid solution to co-precipitate lignin and glucose dehydrogenase. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then freeze-dry to obtain immobilized enzyme.

[0081] Example 6: A method for in-situ encapsulation of enzymes based on lignin

[0082] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0083] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0084] S1. Add KOH solid to 100g of an aqueous dispersion of 0.05% alkali lignin, adjust the pH to 10.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0085] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 0.2g of liquid bromelain containing 50% glycerol (derived from pineapple stem, enzyme activity 100U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0086] S3. Adjust the pH of the mixture to 3.5 with sulfuric acid solution to co-precipitate lignin and bromelain. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then air-dry (40℃) to obtain immobilized enzyme.

[0087] Example 7: A method for in-situ encapsulation of enzymes based on lignin

[0088] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0089] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0090] S1. Add KOH solid to 100g of an aqueous dispersion of 0.1% alkali lignin, adjust the pH to 10.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0091] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 0.2g of liquid bromelain containing 50% glycerol (derived from pineapple stem, enzyme activity 100U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0092] S3. Adjust the pH of the mixture to 3.5 with sulfuric acid solution to co-precipitate lignin and bromelain. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then air-dry (40℃) to obtain immobilized enzyme.

[0093] Example 8: A method for in-situ encapsulation of enzymes based on lignin

[0094] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0095] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0096] S1. Add KOH solid to 100g of an aqueous dispersion of 0.05% alkali lignin, adjust the pH to 14.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0097] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 0.2g of liquid bromelain containing 50% glycerol (derived from pineapple stem, enzyme activity 100U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0098] S3. Adjust the pH of the mixture to 3.5 with sulfuric acid solution to co-precipitate lignin and bromelain. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then air-dry (40℃) to obtain immobilized enzyme.

[0099] Example 9: A method for in-situ encapsulation of enzymes based on lignin

[0100] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0101] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0102] S1. Add KOH solid to 100g of an aqueous dispersion of 0.05% alkali lignin, adjust the pH to 10.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0103] S2. Adjust the pH of the lignin solution to 5.0 with sulfuric acid solution, then add 0.2g of liquid bromelain containing 50% glycerol (derived from pineapple stem, enzyme activity 100U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0104] S3. Adjust the pH of the mixture to 3.5 with sulfuric acid solution to co-precipitate lignin and bromelain. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then air-dry (40℃) to obtain immobilized enzyme.

[0105] Example 10: A method for in-situ encapsulation of enzymes based on lignin

[0106] The lignin used in this embodiment is alkali lignin, which is sourced from Luohe Huadong Lignin Co., Ltd.

[0107] The specific steps for in-situ encapsulation of enzymes using alkali lignin are as follows:

[0108] S1. Add KOH solid to 100g of an aqueous dispersion of 0.05% alkali lignin, adjust the pH to 10.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0109] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 0.2g of liquid bromelain containing 50% glycerol (derived from pineapple stem, enzyme activity 100U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0110] S3. Adjust the pH of the mixture to 4.0 with sulfuric acid solution to co-precipitate lignin and bromelain. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then air-dry (40℃) to obtain immobilized enzyme.

[0111] Example 11: A method for in-situ encapsulation of enzymes based on lignin

[0112] The lignin used in this embodiment was obtained by extracting poplar wood chips at 120°C for 30 minutes using tetrabutylammonium tetrafluoroborate. The extract was diluted with water to precipitate the precipitate, and the precipitate was dried to obtain lignin.

[0113] The specific steps for using this lignin-encapsulated enzyme in situ are as follows:

[0114] S1. Add NaOH solid to 100g of an aqueous dispersion of 0.3% lignin, adjust the pH to 12.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0115] S2. Adjust the pH of the lignin solution to 9.0 with hydrochloric acid solution, then add 0.3g of lipase powder containing 20% ​​starch (derived from Aspergillus oryzae, enzyme activity 300U / mg) to the above system, mix evenly at 24-26℃ to obtain a mixture;

[0116] S3. Adjust the pH of the mixture to 3.2 with hydrochloric acid solution to co-precipitate lignin and lipase. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then freeze-dry to obtain immobilized enzyme.

[0117] Example 12: A method for in-situ encapsulation of enzymes based on lignin

[0118] The lignin used in this embodiment was obtained by extracting birch powder at 120°C for 30 minutes using a solution containing 50% acetylcholine and 50% urea by mass. The extract was diluted with water to precipitate the precipitate, and the precipitate was dried to obtain lignin.

[0119] The specific steps for using this lignin-encapsulated enzyme in situ are as follows:

[0120] S1. Add KOH solid to 100g of an aqueous dispersion of 0.5% lignin, adjust the pH to 11.0, let stand for 30min, filter to remove impurities, and obtain a lignin solution;

[0121] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 1.0g of liquid catalase (derived from Bacillus spp., enzyme activity 80U / mg) containing 70% glycerol by mass to the above system, mix evenly at 24-26℃ to obtain a mixed solution;

[0122] S3. Adjust the pH of the mixture to 3.8 with sulfuric acid solution to co-precipitate lignin and catalase. Separate the precipitate by centrifugation at 8000 rpm for 10 min, and then vacuum dry (40℃) to obtain immobilized enzyme.

[0123] Example 13 Enzyme protein activity test

[0124] I. Experimental Methods

[0125] 1. Enzyme activity test

[0126] (1) Glucose oxidase activity

[0127] Under the action of glucose oxidase, glucose and oxygen react to produce gluconic acid and hydrogen peroxide. Hydrogen peroxide and colorless reduced o-anisidine are reacted with peroxidase to produce water and red oxidized o-anisidine. This red substance has a specific absorption peak at a wavelength of 500 nm. The activity of the enzyme can be calculated by measuring the rate of increase of the absorbance of the red substance at 500 nm.

[0128] Add 2.4 mL of 0.21 mol / L o-anisidine, 0.5 mL of 1 g / L glucose solution, and 0.1 mL of 0.1 g / L horseradish peroxidase to separate test tubes. Shake well and incubate at 35°C for 5 min. Then, add 0.1 mL of sample for detection and record the absorbance (A) every 30 s at a wavelength of 500 nm. 500 nm ). With A 500 nm - Plot the time-time curve and find the maximum slope ΔA (min). Calculate the activity of glucose oxidase using the following formula:

[0129]

[0130] In the formula: V1 is the total volume of the reaction solution, mL; V2 is the volume of the added sample solution, mL; 7.5 is the extinction coefficient of oxidized o-anisidine.

[0131] (2) β-glucosidase activity

[0132] β-glucosidase catalyzes the hydrolysis of p-nitrobenzene-β-D-glucopyranoside (pNPG) to produce p-nitrophenol and glucose. p-Nitrophenol is yellow under alkaline conditions and has a characteristic absorption peak at 400 nm. The activity of β-glucosidase can be indirectly reflected by measuring the change in absorbance of the reaction system at 400 nm.

[0133] Add 1 mL of 0.05 mol / L citrate buffer (pH 4.8) and 0.1 mL of crude enzyme solution diluted by a certain factor to a 25 mL test tube, and preheat to 50 °C. Add 0.9 mL of 5 mmol / L pNPG solution and incubate in a 50 °C water bath for 10 min. Quickly add 1 mL of 1 mol / L sodium carbonate solution, add pure water to a final volume of 25 mL, mix well, and measure the absorbance at 410 nm to obtain the p-nitrophenol content. Calculate the β-glucosidase activity using the following formula:

[0134]

[0135] Where m is the p-nitrophenol content (μmol) and n is the dilution factor of the crude enzyme solution.

[0136] One β-glucosidase activity unit (U) is defined as the amount of p-nitrophenol that is produced per minute by hydrolyzing p-nitrophenol-β-D-glucosidase (pNPG) at 50°C and pH 4.8 to generate 1 μmol of p-nitrophenol.

[0137] (3) Ethanol dehydrogenase activity

[0138] In the reaction where alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde, the coenzyme NADH (reduced nicotinamide adenine dinucleotide) is converted to NAD. + (Oxidized nicotinamide adenine dinucleotide) NADH has a characteristic absorption peak at 340 nm. By measuring the rate of decrease in absorbance at 340 nm of the reaction system, the activity of alcohol dehydrogenase can be indirectly reflected.

[0139] In a 5 mL EP tube, add 10 μL of alcohol dehydrogenase enzyme solution (replace the enzyme solution with an equal volume of ultrapure water for zeroing), 990 μL of SPS buffer (0.1 mol / L, pH 8.8), and 1000 μL of coenzyme NAD+. + The solution (2.5 mmol / L) was prepared, and then 1000 μL of substrate EtOH solution (2 mol / L) was added. After shaking to activate the reaction, the absorbance of the solution at 340 nm was measured after 3 min. The enzyme activity was calculated according to the following formula.

[0140]

[0141] In the formula, v is the enzyme activity, μmol / min; ΔA is the absorbance change value; Δt is the reaction time, min; V is the reaction liquid volume, mL; 1 is the optical path length, cm; and 6.22 is the molar absorptivity of NADH, L / (mol·cm).

[0142] (4) Glucose isomerase activity

[0143] Glucose isomerase can catalyze the conversion of glucose into fructose. During the reaction, carbazole reagent can be used to react with fructose to produce a colorimetric reaction. The activity of the enzyme can be reflected by measuring the change in absorbance of the reaction system at a specific wavelength.

[0144] After diluting the crude enzyme solution appropriately, take 100 μL and add 250 μL of 0.6 M D-glucose and 200 μL of 0.025 M phosphate buffer sequentially, then shake well. Incubate at 70 °C for 15 min, then add 50 μL of 50% trichloroacetic acid solution to terminate the reaction. Immediately afterward, add 3 mL of 70% sulfuric acid solution, 100 μL of 2.4% cysteine ​​hydrochloride solution, and 100 μL of 0.12% ethanol-carbazole solution. Shake well again; the solution will now be a pale purple color. Quickly incubate the reaction system at 60 °C for 30 min to allow the fructose color reaction to fully occur. Remove the tube and cool it in an ice bath; the solution will then turn a deep purple color.

[0145] Using a UV-Vis spectrophotometer, the colorimetric solution was appropriately diluted and placed in a cuvette. The absorbance was measured at 560 nm, with a blank control containing 50 μL of 50% trichloroacetic acid added before the reaction. The measured absorbance was then substituted into the fructose standard curve and multiplied by the dilution factor to obtain the fructose content produced in the reaction.

[0146] The enzyme activity unit of glucose isomerase is defined as: under the above reaction conditions, the amount of enzyme that produces 1 μg of fructose per minute is defined as 1 enzyme activity unit (U).

[0147] (5) Glucose dehydrogenase activity

[0148] Glucose dehydrogenase can catalyze the reaction of D-glucose and NAD+. + The reaction produces D-gluconic acid and NADH, and NADH has a characteristic absorption peak at 340 nm. By measuring the change in absorbance of the reaction system at 340 nm, the activity of glucose dehydrogenase can be indirectly reflected.

[0149] The standard reaction solution for glucose dehydrogenase activity assay consists of 100 mM phosphate buffer (pH 8.0), 200 mM glucose, and 1 mM NAD. +The total volume was 1 mL. 1 μL of enzyme solution of a certain dilution was added to the standard reaction solution, and the mixture was incubated at 25 °C for 5 min. The absorbance was measured at 340 nm. A standard curve was plotted with the concentration of the NADH standard solution on the x-axis and the absorbance on the y-axis. The amount of NADH produced was calculated from the standard curve. The absorptivity of NADH was 6.2 × 10⁻⁶ L / (mol·cm).

[0150] One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of NADH per minute under the given conditions. All measurements were repeated three times. Protein concentration was determined using the Bradford method, with BSA as the standard.

[0151] (6) Bromelain enzyme activity

[0152] Enzyme activity assay: Accurately measure 500 μL of bromelain solution or weigh an appropriate amount of immobilized bromelain into a stoppered test tube. Preheat the enzyme solution and casein solution separately at a test temperature of 37 ± 0.5℃ and pH = 7.0 ± 0.5 for 5 min. Then, add 1000 μL of casein solution to the stoppered test tube containing the bromelain enzyme solution, gently shake to mix, and react precisely at the above temperature for 10 min. Add 1000 μL of trichloroacetic acid solution, shake to mix, and let stand at 37 ± 0.5℃ for 40 min. Remove and cool to room temperature, then centrifuge at 5000 rpm for 10 min. Take the supernatant and measure its absorbance (A) at 275 nm wavelength within 2 h.

[0153] Blank test: Accurately measure 500 μL of bromelain solution or weigh an appropriate amount of immobilized bromelain into a stoppered test tube, add 1000 μL of trichloroacetic acid solution, shake gently, and react precisely for 10 min under the same temperature conditions. Then add 1000 μL of casein solution, shake well, and let stand at 37 ± 0.5 °C for 40 min. Remove and cool to room temperature, then centrifuge at 5000 rpm for 10 min. Take the supernatant and measure its absorbance A0 at 275 nm within 2 h.

[0154] Bromelain activity is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute under the test conditions (37±5℃, pH=7.0), which is defined as one enzyme activity unit (U).

[0155]

[0156] In the formula, A is the absorbance value of the sample solution at 275 nm; A0 is the absorbance value of the blank solution at 275 nm; K is obtained from the tyrosine standard curve; V is the volume of the reaction solution (mL); t is the reaction time (min); m is the enzyme content in the reaction system (mg); and n is the dilution factor.

[0157] (7) Lipase activity

[0158] Prepare 1.5 mL of Tri-HCl buffer (pH 8.0, 100 mM), ensuring the enzyme concentration is 0.1 mg / mL, and add the substrate p-nitrophenyl hexanoate to a final concentration of 10 mM. Incubate the system at 50 °C for 1.5 h. After incubation, cool the system for 10 min to terminate the reaction, and measure the absorbance of p-nitrophenol at 410 nm using a spectrophotometer. The measured value should be used to calculate the amount of p-nitrophenol by referring to a p-NP standard curve established under the same conditions (with p-NP concentration as the x-axis and absorbance at 410 nm as the y-axis). When measuring enzyme activity, the amount of free enzyme added should be equal to the amount of enzyme encapsulated in the enzyme complex.

[0159] Lipase activity (U) is defined as the amount of enzyme required to catalyze the hydrolysis of a substrate to produce 1.0 μmol of p-nitrophenol by 1 mg of free / immobilized enzyme at 50°C and pH 8.0 for 1 min. The formula for lipase activity is as follows:

[0160]

[0161] Where X represents lipase activity (U); c represents p-NP concentration (μmol·L⁻¹). -1 V is the final volume of the reaction solution (L); t is the reaction time (min); m is the amount of enzyme used (mg).

[0162] (8) Catalase activity

[0163] Hydrogen peroxide (H₂O₂) has strong absorption at a wavelength of 240 nm, and catalase can decompose hydrogen peroxide, thus increasing the absorbance of the reaction solution (A). 240 The absorbance decreases with reaction time. The activity of catalase can be determined by measuring the rate of change in absorbance. At room temperature, 0.3 mL of catalase solution was added to 9.7 mL of 20 mmol / L H₂O₂ solution, quickly mixed, and poured into a quartz cuvette. The absorbance was measured at 240 nm using a UV-Vis spectrophotometer, with readings taken every 0.5 min for a total of 2 min.

[0164] 2. Encapsulation efficiency of enzyme proteins

[0165] In this embodiment, the encapsulation rate of the enzyme protein was obtained by testing the protein content in the supernatant after co-precipitation of lignin and enzyme using UV-Vis spectrophotometry. After co-precipitation of lignin and enzyme, centrifugation was performed, and the supernatant was poured into a quartz cuvette. The absorbance was measured using a UV-Vis spectrophotometer. By comparing the absorbance with that of pure lignin, the encapsulation rate of the enzyme protein could be obtained.

[0166] 3. Immobilized enzyme loading rate

[0167] The loading rate of the immobilized enzyme was calculated by converting the nitrogen content through elemental analysis. Weigh (5.00±0.25) mg of sample, wrap it firmly in aluminum foil, and place it in a sample tray. Turn on the instrument, gas valve, and computer connected to the elemental analyzer. Set the program for automatic detection. The computer automatically calculates the protein content of the sample based on the calibration curve of the standard substance. Each sample is tested in triplicate, with three control tests using L-cysteine ​​as the reference substance. The average value of the test results is the protein content of the sample, and the coefficient of variation (RSD) is calculated. The nitrogen content of both the free enzyme and the immobilized enzyme is measured separately, and the loading rate of the immobilized enzyme is calculated using the following formula.

[0168]

[0169] 4. Enzyme activity retention rate

[0170] Enzyme activity retention rate is obtained by comparing the activities of immobilized and free enzymes under the same protein mass. The enzyme activities of immobilized and free enzymes are measured according to enzyme activity assay methods, and the enzyme activity retention rate is calculated using the following formula.

[0171]

[0172] II. Experimental Results

[0173] The results are shown in Table 1. Table 1 shows that the encapsulation rates of the immobilized enzymes prepared in Examples 1-12 all exceeded 80%, with most reaching over 90%. The encapsulation rate of bromelain in Example 6 was relatively low, mainly due to the low initial ratio of lignin to enzyme protein (1:2), which led to saturation of enzyme protein adsorption on the lignin, thus reducing the encapsulation rate. The enzyme loading rate is mainly related to the initial mass ratio of lignin to enzyme and the encapsulation rate, and can be adjusted according to specific application conditions.

[0174] The immobilized enzymes prepared in Examples 1-12 all retained more than 70% of the activity of the free enzyme. In Example 11, the activity of the lipase even exceeded that of the free enzyme. This may be because lignin has a certain improving effect on the structure of the lipase.

[0175] Table 1. Effects of Immobilized Enzymes

[0176] Example enzymes Packaging ratio Enzyme loading rate Immobilized enzyme activity Enzyme activity retention rate 1 glucose oxidase 90％ 45％ 78U / mg 78％ 2 β-glucosidase 94％ 21％ 9U / mg 75％ 3 alcohol dehydrogenase 98％ 15.8％ 300U / mg 96.8％ 4 Glucose isomerase 91％ 30％ 27U / mg 90％ 5 glucose dehydrogenase 92％ 18.5％ 210U / mg 91.3％ 6 Bromelain 82％ 61％ 84U / mg 84％ 7 Bromelain 90％ 47％ 82U / mg 82％ 8 Bromelain 84％ 62.7％ 72U / mg 72％ 9 Bromelain 62％ 55.4％ 83U / mg 83％ 10 Bromelain 52％ 53.7％ 85U / mg 85％ 11 Lipase 90％ 40％ 312U / mg 104％ 12 catalase 94％ 34％ 72U / mg 90％

[0177] Example 14 Characterization of immobilized enzymes

[0178] I. Experimental Methods

[0179] Scanning electron microscopy combined with energy-dispersive X-ray ablation (SEM-EDXA) was used. The surface morphology of the immobilized β-glucosidase in Example 2 was observed using an electron microscope (Zeiss Sigma 300), and the elemental distribution of the immobilized β-glucosidase was characterized by SEM-mapping using energy-dispersive X-ray ablation (EDXA). Before observation, the sample was freeze-dried and then observed using a scanning electron microscope. Prior to observation, a small amount of sample was adhered to conductive adhesive and sputtered with gold for 45 seconds. The surface morphology of the sample was observed under high vacuum conditions, with an accelerating voltage of 200V–30kV and an electron beam current range of 0.3Pa–100nA.

[0180] II. Experimental Results

[0181] Lignin exhibits an irregular blocky structure, with a uniform distribution on its surface. Figure 1 In the elemental distribution map, carbon (C) dominates at 66.14%, followed by oxygen (O) at 28.84%, while nitrogen (N) and sulfur (S) have lower contents, at 0.52% and 0.36% respectively (Table 2), which is consistent with the chemical properties of lignin.

[0182] The SEM images of the immobilized enzyme showed a coarse and irregular aggregated structure. Figure 2 This is significantly different from the morphology of blocky lignin, possibly because the addition of the enzyme affected the relatively regular self-assembly of lignin. Mapping elemental analysis showed that the nitrogen content of the immobilized enzyme increased significantly from 0.52% to 7.41%. Figure 3 (See Table 2). This change indicates that the addition of enzymes significantly increased the N content in the lignin complex. Observation of the N element distribution shows that during the enzyme encapsulation process, the enzyme was relatively evenly distributed (as shown in Table 2). Figure 3 ).

[0183] Table 2. Spectrum of elemental distribution in lignin samples.

[0184] element Line type wt% Wt%Sigma At% C K-line system 66.14 0.33 73.05 N K-line system 0.52 0.43 0.50 O K-line system 28.84 0.21 23.92 Na K-line system 4.14 0.06 2.39 S K-line system 0.36 0.04 0.15 Total 100.00 100.00

[0185] Table 3. Elemental distribution spectrum of immobilized enzyme samples

[0186] element Line type wt% Wt%Sigma At% C K-line system 67.66 0.22 73.15 N K-line system 7.41 0.27 6.87 O K-line system 24.20 0.13 19.64 Na K-line system 0.22 0.02 0.13 S K-line system 0.51 0.02 0.21 Total 100.00 100.00

[0187] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for encapsulating enzymes in situ with lignin, characterized by, Includes the following steps: S1. Disperse industrial lignin in water to obtain a dispersion, adjust the pH of the dispersion to above 10.0, mix thoroughly and remove insoluble matter to obtain an industrial lignin solution; S2. Adjust the pH of the industrial lignin solution to 5-9.5, then add enzyme protein, mix thoroughly to obtain a mixture; S3. Adjust the pH of the mixture to 3.0-4.0 to co-precipitate industrial lignin and enzyme protein, then separate the precipitate and dry it to obtain the final product; The enzyme protein is one or more of the following: glucose oxidase from Aspergillus niger, β-glucosidase from almond, alcohol dehydrogenase from Saccharomyces cerevisiae, glucose isomerase from Lactobacillus, glucose dehydrogenase from Pseudomonas, bromelain from pineapple stem, lipase from Aspergillus oryzae, and catalase from Bacillus. The industrial lignin is one or more of alkali lignin, enzymatically hydrolyzed lignin, ethanol lignin, and lignin extracted from plant raw materials using ionic liquids, deep eutectic solvents, or hydrated molten salt solvents.

2. The method of claim 1, wherein, In step S1, the mass fraction of industrial lignin in the dispersion is 0.05-2%.

3. The method of claim 1, wherein, In step S1, thorough mixing means thorough mixing at 24–26°C.

4. The method of claim 1, wherein, The mass of the enzyme protein is 20-200% of the mass of industrial lignin.

5. The method of claim 1, wherein, The enzyme protein contains a protective agent; The enzyme protein is glucose oxidase derived from Aspergillus niger, and the protectant is maltodextrin. The enzyme protein is β-glucosidase from almond, alcohol dehydrogenase from Saccharomyces cerevisiae, bromelain from pineapple stem, or catalase from Bacillus subtilis, and the protectant is glycerol. The enzyme protein is glucose dehydrogenase derived from Pseudomonas, and the protective agent is carboxymethyl cellulose; The enzyme protein is a glucose isomerase derived from Lactobacillus or a lipase derived from Aspergillus oryzae, and the protectant is starch.

6. The method of claim 5, wherein, The mass of the protective agent is 20-400% of the enzyme protein mass.

7. The immobilized enzyme prepared by the method according to any one of claims 1 to 6.

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