Method for in-situ encapsulation of enzyme by using lignin

Abstract

The invention discloses a method for in-situ encapsulation of enzyme by using lignin. The method comprises the following steps: adjusting the pH value of an industrial lignin aqueous dispersion to 10.0 or above to dissolve industrial lignin, filtering to remove impurities to obtain an industrial lignin solution, then adjusting the pH value of the industrial lignin solution to 5-9.5, adding zymoprotein to obtain a mixed solution, adjusting the pH value of the mixed solution to 3.0-4.0 again to co-precipitate the industrial lignin and the zymoprotein, and carrying out solid-liquid separation to obtain a solid product. And separating the precipitate, and drying to obtain the immobilized enzyme. According to the method, the enzyme with low pH sensitivity is immobilized by utilizing the adsorption capacity of lignin to the enzyme and the property of alkali dissolution and acidification of lignin, so that not only is the encapsulation rate of the enzyme improved, but also the activity of the immobilized enzyme is better reserved. The invention provides a novel zymoprotein in-situ packaging method which has positive significance in promoting the development of lignocellulose biorefinery and green biological manufacturing.

Description

Technical Field

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

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

[0003] Currently, common enzyme immobilization methods mainly include physical adsorption method, covalent binding method, cross-linking method, and embedding method, etc. The physical adsorption method is simple to operate, but the binding force is weak and the enzyme is easy to fall off; the covalent binding method has a strong binding, but the reaction conditions are relatively harsh and may affect the enzyme activity; although the cross-linking method can improve the enzyme stability, it is also easy to cause a decrease in enzyme activity; the embedding method can better maintain the enzyme activity, but traditional embedding materials often have disadvantages such as large mass transfer resistance, low mechanical strength, and high cost.

[0004] Among the existing enzyme immobilization methods, it is rare to simultaneously meet the requirements such as high stability, high activity, easy recycling, and environmental friendliness. In addition, most of the existing embedding materials are synthetic materials, such as artificial polymers, metal-organic frameworks, covalent organic frameworks, etc., whose preparation processes are complex, the costs are high, and they may cause pollution to the environment.

[0005] To solve the problems of enzyme immobilization, different methods have been proposed in the prior art. Chinese Patent CN1810867A discloses a method for embedding enzyme molecules using sodium alginate / chitosan hydrogel. This method improves the enzyme stability to a certain extent, but the water-locking property of the hydrogel easily leads to a large mass transfer barrier between the enzyme and the substrate, and the mechanical properties of the hydrogel are poor, making industrial application difficult. Chinese Patent CN117700696A discloses a method for encapsulating enzyme molecules using polylactic acid microspheres. Although the stability of the enzyme molecules is significantly improved, organic solvents are used in the preparation process of the polylactic acid microspheres, and the organic solvents need to be removed by long-term volatilization, which is not suitable for different enzymes and is difficult to promote and apply.

[0006] In summary, there is an urgent need for a simple and efficient enzyme immobilization method that can overcome the deficiencies of the existing methods, improve the stability, activity, and recycling rate of enzymes, and at the same time has the advantages of environmental friendliness, simple preparation, and low cost. Summary of the Invention

[0007] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a method for in-situ encapsulating enzymes using lignin.

[0008] The first object of the present invention is to provide a method for in-situ encapsulating enzymes using lignin.

[0009] The second object of the present invention is to provide an immobilized enzyme prepared by the above method.

[0010] The present invention claims the following:

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

[0012] S1. Dispersing industrial lignin in water to obtain a dispersion, adjusting the pH value of the dispersion to above 10.0, fully mixing and removing insoluble substances to obtain an industrial lignin solution;

[0013] S2. Adjusting the pH value of the industrial lignin solution to 5 - 9.5, then adding enzyme protein, and fully mixing to obtain a mixture;

[0014] S3. Adjusting the pH value of the mixture to 3.0 - 4.0, causing the industrial lignin and the enzyme protein to co-precipitate, then separating the precipitate and drying to obtain the product.

[0015] As an implementable mode, the industrial lignin is one or several of alkali lignin, enzymatic hydrolysis lignin, organic solvent lignin, Klason lignin, and lignin extracted from plant raw 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 full mixing is carried out under the condition of 24 - 26 °C.

[0018] As an implementable mode, in step S1, an alkali solution prepared from one or several of NaOH, KOH, Ca(OH) 2 and ammonia water is used to adjust the pH.

[0019] Preferably, in step S2, the enzyme protein is one or several of glucose oxidase, β-glucosidase, alcohol dehydrogenase, glucose isomerase, glucose dehydrogenase, bromelain, lipase, catalase, protease, transaminase, glycosyltransferase, 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 in-situ encapsulating enzymes with lignin can be guaranteed to be ≥90%.

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

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

[0023] S1. Disperse the alkali lignin in water to obtain a dispersion, where 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, fully mix and then remove the insoluble matter to obtain an alkali lignin solution;

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

[0025] S3. Adjust the pH value of the mixed solution to 3.5 to cause the co-precipitation of alkali lignin and enzyme protein, then separate the precipitate, and dry it to obtain the product;

[0026] In this preferred scheme, the encapsulation rate of using alkali lignin to encapsulate bromelain 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, and the enzyme protein is bromelain. The method includes the following steps:

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

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

[0031] S3. Adjust the pH value of the mixed solution to 3.5 to cause the co-precipitation of alkali lignin and enzyme protein, then separate the precipitate, and dry it to obtain the product;

[0032] In this preferred scheme, the loading rate of using alkali lignin to encapsulate bromelain can reach 61 - 63%.

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

[0034] S1. Disperse alkali lignin in water to obtain a dispersion, where 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 well and remove insoluble substances to obtain an alkali lignin solution;

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

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

[0037] Under this preferred scheme, the retention rate of the enzyme activity of bromelain encapsulated by alkali lignin changes little and can reach 82 - 85%.

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

[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 several 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 mass of the enzyme protein.

[0043] The immobilized enzyme prepared by any of the above - mentioned methods.

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

[0045] The present invention discloses a method for in - situ encapsulating an enzyme using lignin. The present invention utilizes the adsorption ability of lignin to the enzyme and its property of alkali - soluble and acid - precipitated with low sensitivity to pH to immobilize enzymes with low sensitivity to pH, which not only improves the encapsulation rate of the enzyme but also better retains the activity of the immobilized enzyme. The retention rate of the enzyme activity after immobilization reaches 72 - 104%. At the same time, the method of the present invention is green, environmentally friendly, simple, efficient, and easy to promote and apply. The present invention provides a new method for in - situ encapsulation of enzyme protein, which has a positive significance for promoting the development of lignocellulosic biorefining and green biomanufacturing. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0047] Figure 2 It is the scanning electron microscope image of the immobilized enzyme sample.

[0048] Figure 3 It is the scanning electron microscope image of the N element distribution of the immobilized enzyme sample. Specific embodiments

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

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

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

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

[0053] The specific steps for in-situ encapsulation of enzyme using Longli enzymatically hydrolyzed lignin are as follows:

[0054] S1. Add NaOH powder to 100 g of an aqueous dispersion of Longli enzymatically hydrolyzed lignin with a mass fraction of 0.1%, adjust the pH to 12.0, let it stand for 30 min, and then filter to remove impurities to obtain a lignin solution.

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

[0056] S3. Adjust the pH of the mixed solution to 3.0 with hydrochloric acid to cause co-precipitation of lignin and glucose oxidase, centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform freeze-drying to obtain the immobilized enzyme.

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

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

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

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

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

[0062] S3. Adjust the pH of the mixed solution to 3.5 with sulfuric acid solution to co-precipitate lignin and β-glucosidase. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform freeze-drying to obtain the immobilized enzyme.

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

[0064] The lignin used in this example is Longli enzymatic hydrolysis lignin, which is derived from Shandong Longli Biotechnology Co., Ltd. and is extracted from the residue after preparing functional sugars from corncobs.

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

[0066] S1. Add solid KOH to a 100 g aqueous dispersion of Longli enzymatic hydrolysis lignin with a mass fraction of 0.2% to adjust the pH to 12.5. After standing for 30 min, filter to remove impurities to obtain the lignin solution.

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

[0068] S3. Adjust the pH of the mixed solution to 3.5 with sulfuric acid solution to co-precipitate lignin and alcohol dehydrogenase. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform freeze-drying to obtain the immobilized enzyme.

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

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

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

[0072] S1. Add Ca(OH) 2 solid to a 100 g aqueous dispersion of alkali lignin with a mass fraction of 1% to adjust the pH to 11.0. After standing for 30 min, filter to remove impurities to obtain the 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 by mass (derived from Lactobacillus, enzyme activity 30 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0074] S3. Adjust the pH of the mixed solution to 4.0 with phosphoric acid solution to co-precipitate lignin and glucose isomerase. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and vacuum dry at 40 °C to obtain the immobilized enzyme.

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

[0076] The lignin used in this example is ethanol lignin, extracted from corn stover, and obtained by extracting with 60% ethanol aqueous solution at 160 °C for 30 min.

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

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

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

[0080] S3. Adjust the pH of the mixed solution to 3.2 with sulfuric acid solution to co-precipitate lignin and glucose dehydrogenase. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform freeze-drying to obtain the immobilized enzyme.

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

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

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

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

[0085] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 0.2 g of liquid bromelain containing 50% glycerol by mass (derived from pineapple stems, enzyme activity 100 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0086] S3. Adjust the pH of the mixed solution to 3.5 with sulfuric acid solution to cause co - precipitation of lignin and bromelain. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform air drying (40 °C) to obtain the immobilized enzyme.

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

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

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

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

[0091] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, then add 0.2 g of liquid bromelain containing 50% glycerol by mass (derived from pineapple stems, enzyme activity 100 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0092] S3. Adjust the pH of the mixed solution to 3.5 with sulfuric acid solution to cause co - precipitation of lignin and bromelain. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform air drying (40 °C) to obtain the immobilized enzyme.

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

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

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

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

[0097] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, and then add 0.2 g of liquid bromelain containing 50% glycerol by mass (derived from pineapple stems, enzyme activity 100 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0098] S3. Adjust the pH of the mixed solution to 3.5 with sulfuric acid solution to co-precipitate lignin and bromelain. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform air drying (40 °C) to obtain the immobilized enzyme.

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

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

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

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

[0103] S2. Adjust the pH of the lignin solution to 5.0 with sulfuric acid solution, and then add 0.2 g of liquid bromelain containing 50% glycerol by mass (derived from pineapple stems, enzyme activity 100 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0104] S3. Adjust the pH of the mixed solution to 3.5 with sulfuric acid solution to co-precipitate lignin and bromelain. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform air drying (40 °C) to obtain the immobilized enzyme.

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

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

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

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

[0109] S2. Adjust the pH of the lignin solution to 7.0 with a sulfuric acid solution, then add 0.2 g of liquid bromelain containing 50% glycerol by mass (derived from pineapple stems, enzyme activity 100 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0110] S3. Adjust the pH of the mixed solution to 4.0 with a sulfuric acid solution to co-precipitate lignin and bromelain. Centrifuge the precipitate at 8000 rpm for 10 min to separate the precipitate, and then perform air drying (40 °C) to obtain the immobilized enzyme.

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

[0112] The lignin used in this example was extracted from poplar chips at 120 °C for 30 min using tetrabutylammonium tetrafluoroborate. The extract was diluted with water to cause precipitation, and the precipitate was dried to obtain lignin.

[0113] The specific steps for in-situ encapsulation of enzymes using this lignin are as follows:

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

[0115] S2. Adjust the pH of the lignin solution to 9.0 with a hydrochloric acid solution, then add 0.3 g of lipase powder containing 20% starch by mass (derived from Aspergillus oryzae, enzyme activity 300 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained;

[0116] S3. Adjust the pH of the mixed solution to 3.2 with a hydrochloric acid solution to co-precipitate lignin and lipase. Centrifuge the precipitate at 8000 rpm for 10 min to separate the precipitate, and then perform freeze-drying to obtain the immobilized enzyme.

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

[0118] The lignin used in this example was extracted from birch powder at 120 °C for 30 min using a solution containing 50% acetylcholine and 50% urea by mass. The extract was diluted with water to cause precipitation, and the precipitate was dried to obtain lignin.

[0119] The specific steps for in-situ encapsulation of enzymes using this lignin are as follows:

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

[0121] S2. Adjust the pH of the lignin solution to 7.0 with sulfuric acid solution, and then add 1.0 g of liquid hydrogen peroxidase containing 70% by mass of glycerol (derived from the genus Bacillus, enzyme activity 80 U / mg) to the above system. After mixing evenly at 24 - 26 °C, a mixed solution is obtained.

[0122] S3. Adjust the pH of the mixed solution to 3.8 with sulfuric acid solution to co-precipitate lignin and hydrogen peroxidase. Centrifuge at 8000 rpm for 10 min to separate the precipitate, and then perform vacuum drying (40 °C) to obtain the immobilized enzyme.

[0123] Example 13 Activity Test of Enzyme Protein

[0124] I. Experimental Method

[0125] 1. Enzyme Activity Test

[0126] (1) Glucose Oxidase Activity

[0127] Under the action of glucose oxidase, glucose and oxygen react to form gluconic acid and hydrogen peroxide. Hydrogen peroxide and colorless reduced o-dianisidine react under the action of peroxidase to form water and red oxidized o-dianisidine. 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 increasing rate of the absorbance of the red substance at 500 nm.

[0128] Add 2.4 mL of 0.21 mol / L o-dianisidine, 0.5 mL of 1 g / L glucose solution, and 0.1 mL of 0.1 g / L horseradish peroxidase into a test tube respectively. After shaking well, incubate in a water bath at 35 °C for 5 min. Then add 0.1 mL of the sample during detection, and record the absorbance (A 500 nm ) at a wavelength of 500 nm every 30 s. Plot A 500 nm - time graph to find the maximum slope △A (min). Calculate the activity of glucose oxidase according to the following formula:

[0129]

[0130] In the formula: V 1 is the total volume of the reaction solution, mL; V 2 is the volume of the added sample solution, mL; 7.5 is the extinction coefficient of oxidized o-dianisidine.

[0131] (2) β-Glucosidase Activity

[0132] β-glucosidase can catalyze the hydrolysis of p-nitrophenyl-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. By measuring the change in absorbance of the reaction system at 400 nm, the activity of β-glucosidase can be indirectly reflected.

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

[0134]

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

[0136] One unit (U) of β-glucosidase activity is defined as: hydrolyzing p-nitrophenyl-β-D-glucoside (pNPG) to produce 1 μmol of p-nitrophenol per minute at 50 °C and pH 4.8.

[0137] (3) Ethanol dehydrogenase activity

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

[0139] In a 5 mL EP tube, sequentially add 10 μL of ethanol dehydrogenase enzyme solution (the zero-adjusting solution uses an equal volume of ultrapure water instead of the enzyme solution), 990 μL of SPS buffer (0.1 mol / L, pH 8.8), 1000 μL of coenzyme NAD + solution (2.5 mmol / L), and then add 1000 μL of substrate EtOH solution (2 mol / L). After shaking to initiate the reaction, start timing and measure the absorbance of the solution at 340 nm at 3 min. Calculate the enzyme activity according to the following formula.

[0140]

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

[0142] (4) Glucose isomerase activity

[0143] Glucose isomerase can catalyze the conversion of glucose to fructose. During the reaction, a color reaction can occur between the carbazole reagent and fructose, and the enzyme activity can be reflected by measuring the change in absorbance of the reaction system at a specific wavelength.

[0144] Dilute the crude enzyme solution by an appropriate multiple, then take 100 μL and add it successively to 250 μL of 0.6 M D-glucose and 200 μL of 0.025 M phosphate buffer, and shake well. Incubate in a 70°C constant temperature water bath for 15 min, then add 50 μL of 50% (v / v) trichloroacetic acid solution to terminate the reaction. Immediately add 3 mL of 70% (v / v) sulfuric acid solution, 100 μL of 2.4% (w / v) cysteine hydrochloride solution, and 100 μL of 0.12% (w / v) ethanol carbazole solution. Shake well again. At this time, the solution shows a light purple color. Quickly incubate the reaction system in a 60°C constant temperature water bath for 30 min to fully carry out the fructose color reaction. Take it out, cool the test tube in an ice bath, and the solution shows a deep purple color.

[0145] Use a UV-visible spectrophotometer. After appropriately diluting the colored solution, place it in a cuvette and measure its absorbance at a wavelength of 560 nm, using the addition of 50 μL of 50% (v / v) trichloroacetic acid before the reaction as a blank control. Substitute the measured absorbance into the fructose standard curve and multiply by the dilution factor to obtain the fructose content generated in the reaction.

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

[0147] (5) Glucose dehydrogenase activity

[0148] Glucose dehydrogenase can catalyze D-glucose and NAD + to generate 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 detecting glucose dehydrogenase activity consists of 100 mM phosphate buffer (pH 8.0), 200 mM glucose, 1 mM NAD +, with a total volume of 1 mL. Add 1 μL of enzyme solution at a certain dilution to the standard reaction solution and incubate at 25 °C for 5 min. Measure the absorbance at 340 nm. Plot a standard curve with the concentration of the NADH standard solution on the abscissa and the absorbance on the ordinate, and calculate the amount of NADH produced in the reaction from the standard curve. The extinction coefficient of NADH is 6.2×10 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 these conditions. All determinations were repeated three times. The protein concentration was measured by the Bradford method using BSA as the standard.

[0151] (6) Bromelain enzyme activity

[0152] Enzyme activity determination: 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 the casein solution at the measurement temperature of 37 ± 0.5 °C and pH = 7.0 ± 0.5 for 5 min. Then, add 1000 μL of the casein solution to the stoppered test tube containing the bromelain enzyme solution, gently shake to mix, and accurately react at the above temperature for 10 min. Add 1000 μL of trichloroacetic acid solution, shake well, and let stand at 37 ± 0.5 °C for 40 min. Take out and cool to room temperature, and centrifuge at 5000 rpm for 10 min. Take the supernatant and measure its absorbance A at a wavelength of 275 nm 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, gently shake, and accurately react at the same temperature for 10 min. Then, add 1000 μL of the casein solution, shake well, let stand at 37 ± 0.5 °C for 40 min, take out and cool to room temperature, and centrifuge at 5000 rpm for 10 min. Take the supernatant and measure its absorbance A at a wavelength of 275 nm within 2 h 0 .

[0154] Definition of bromelain activity: Under the measurement conditions (37 ± 5 °C, pH = 7.0), the amount of enzyme required for bromelain to hydrolyze casein to produce 1 μg of tyrosine per minute is defined as one unit of enzyme activity (U).

[0155]

[0156] Where A is the absorbance value of the sample solution at 275 nm; A 0is 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); n is the dilution factor.

[0157] (7) Lipase enzyme activity

[0158] Prepare 1.5 mL of Tri-HCl buffer (pH 8.0, 100 mM), ensure the enzyme concentration is 0.1 mg / mL, and add the substrate p-nitrophenyl caproate to 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 needs to be used to calculate the amount of p-nitrophenol by referring to the p-NP standard curve established under the same conditions (with the p-NP concentration on the x-axis and the absorbance at 410 nm on the y-axis). When measuring the enzyme activity, the amount of free enzyme added should be equal to the amount of enzyme encapsulated in the enzyme complex.

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

[0160]

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

[0162] (8) Catalase enzyme activity

[0163] Hydrogen peroxide (H 2 O 2 ) has a strong absorption at a wavelength of 240 nm, and catalase can decompose hydrogen peroxide, causing the absorbance (A 240 ) of the reaction solution to decrease with the reaction time. The activity of catalase can be measured according to the change rate of the measured absorbance. At room temperature, add 0.3 mL of catalase solution to 9.7 mL of a reaction system of H 2 O 2 solution with a concentration of 20 mmol / L, mix quickly, pour it into a quartz cuvette, and measure the absorbance at a wavelength of 240 nm using an ultraviolet-visible spectrophotometer. Read the value once every 0.5 min for a total of 2 min.

[0164] 2. Encapsulation rate of enzyme protein

[0165] In the examples, the encapsulation rate of the enzyme protein was obtained by measuring the protein content in the supernatant after co - precipitation of lignin and the enzyme using ultraviolet - visible spectrophotometry. After co - precipitation of lignin and the enzyme, centrifugation was carried out for separation. The supernatant was poured into a quartz cuvette, and the absorbance was measured using an ultraviolet - visible spectrophotometer. By comparing the absorbance of pure lignin, the encapsulation rate of the enzyme protein could be obtained.

[0166] 3. Loading rate of immobilized enzyme

[0167] The loading rate of the immobilized enzyme was obtained by converting the measured N - element content through elemental analysis. Weigh (5.00 ± 0.25) mg of the sample, wrap it with tin foil and compact it, then place it on the sample tray. Turn on the instrument, the gas valve, and the computer connected to the elemental analyzer, set the program, and it can automatically detect. The computer automatically calculates the protein content of the sample to be measured according to the calibration curve of the reference substance. Each sample was repeated 3 times, and 3 control detections were made using L - cystine as the reference substance. The average value of the detection results was the protein content of the sample, and finally its coefficient of variation (RSD) was calculated. The N - element of the free enzyme and the immobilized enzyme was detected respectively, and the loading rate of the immobilized enzyme was calculated according to the following formula.

[0168]

[0169] 4. Retention rate of enzyme activity

[0170] The retention rate of enzyme activity was obtained by comparing the activities of the immobilized enzyme and the free enzyme under the same protein mass. According to the enzyme activity assay method, the activities of the immobilized enzyme and the free enzyme were measured, and the retention rate of enzyme activity was calculated according to the following formula.

[0171]

[0172] II. Experimental results

[0173] The results are shown in Table 1. It can be seen from Table 1 that the encapsulation rates of the immobilized enzymes prepared in Examples 1 - 12 all exceeded 80%, and most of the encapsulation rates reached more than 90%. Among them, the encapsulation rate of bromelain in Example 6 was relatively low, mainly because the initial mass ratio of lignin to enzyme protein was relatively small (1:2), which led to the adsorption of enzyme protein on lignin reaching saturation, so the encapsulation rate decreased. The loading rate of the enzyme is mainly related to the initial mass ratio of lignin and enzyme and the encapsulation rate, and can be adjusted according to the specific application situation.

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

[0175] Table 1 Effects of immobilized enzymes

[0176] Example Enzyme Encapsulation efficiency Loading rate of enzyme Immobilized enzyme activity Retention rate of enzyme activity 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 Enzyme

[0178] I. Experimental Methods

[0179] Scanning electron microscopy combined with energy-dispersive X-ray analysis (SEM-EDXA). The surface morphology of the immobilized β-glucosidase in Example 2 was observed by an electron microscope (Zeiss sigma300), and combined with energy-dispersive X-ray analysis (EDXA) technology, the SEM-mapping elemental distribution characterization of the immobilized β-glucosidase was realized. Before observation, the sample was freeze-dried and then observed by scanning electron microscopy. Before observation, a small amount of the sample was stuck on the conductive adhesive and sputtered with gold for 45 s. The surface morphology of the sample was observed under the conditions of high vacuum mode, acceleration voltage of 200 V - 30 kV, and electron beam current range of 0.3 Pa - 100 nA.

[0180] II. Experimental Results

[0181] Lignin has an irregular block structure, and the distribution on the surface of lignin is uniform ( Figure 1 ). In the Mapping elemental distribution map, carbon (C) dominates with 66.14%, followed by oxygen (O) at 28.84%, and the contents of nitrogen (N) and sulfur (S) are relatively low, 0.52% and 0.36% respectively (Table 2), which is consistent with the chemical properties of lignin.

[0182] The SEM image of the immobilized enzyme shows a rough and irregular agglomerated structure ( Figure 2 ), which is significantly different from the morphology of the massive lignin, probably because the addition of the enzyme affects the relatively regular self-assembly of lignin. The Mapping elemental analysis shows that the N content of the immobilized enzyme increases significantly from 0.52% to 7.41% ( Figure 3 and Table 2). This change indicates that the addition of the enzyme significantly increases the N content in the lignin complex. By observing the distribution of the N element, it can be seen that during the process of lignin encapsulating the enzyme, the enzyme is relatively evenly distributed ( Figure 3 ).

[0183] Table 2 Total elemental distribution spectrogram of lignin samples

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

[0185] Table 3 Total elemental distribution spectrogram of immobilized enzyme samples

[0186] Element Line type wt% Wt%Sigma At% C K series 67.66 0.22 73.15 N K series 7.41 0.27 6.87 O K series 24.20 0.13 19.64 Na K series 0.22 0.02 0.13 S K series 0.51 0.02 0.21 Total amount 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for in situ encapsulation of enzymes using lignin, characterized in that: The following steps are involved: S1. Dispersing industrial lignin in water to obtain a dispersion, adjusting the pH value of the dispersion to above 10.0, mixing thoroughly and removing insoluble matter to obtain an industrial lignin solution; S2. The pH value of the industrial lignin solution is adjusted to 5 to 9.5, and then the enzyme protein is added and mixed thoroughly to obtain a mixed solution; S3. The pH value of the mixed solution is adjusted to 3.0-4.0 to allow the industrial lignin and the enzyme protein to co-precipitate, and then the precipitate is separated and dried to obtain the product.

2. The method according to claim 1, characterized in that The industrial lignin is one or more of alkali lignin, enzymatic lignin, organic solvent lignin, Klason lignin, and lignin extracted from plant raw materials using ionic liquids, deep eutectic solvents or hydrated molten salt solvents.

3. The method according to claim 1, characterized in that In step S1, the mass fraction of industrial lignin in the dispersion is 0.05-2%.

4. The method according to claim 1, characterized in that: In step S1, the sufficient mixing is sufficient mixing at 24-26°C.

5. The method according to claim 1, characterized in that In step S2, the enzyme protein is one or more of glucose oxidase, β-glucosidase, alcohol dehydrogenase, glucose isomerase, glucose dehydrogenase, bromelain, lipase and catalase.

6. The method according to claim 1, characterized in that The mass of the enzyme protein is 20-200% of the mass of industrial lignin.

7. The method according to claim 1, characterized in that The enzyme protein contains a protective agent.

8. The method according to claim 7, characterized in that The protective agent is one or more of carboxymethyl cellulose, starch, maltodextrin, cyclodextrin, diatomaceous earth, glycerol, glucose, polyethylene glycol, polyvinyl pyrrolidone and polyvinyl alcohol.

9. The method according to claim 7, characterized in that: The mass of the protective agent is 20 to 400% of the mass of the enzyme protein.

10. The immobilized enzyme prepared by the method according to any one of claims 1 to 9.

Images