Method for constructing double-enzyme immobilized complete cycle system by utilizing sargassum

Abstract

The invention discloses a method for constructing a double-enzyme immobilized complete circulation system by utilizing gulfweed, and belongs to the technical field of resource utilization of gulfweed biomass cellulose. Enzymolysis residues are fired into a biochar carrier for immobilizing enzyme, and the biochar carrier is used for immobilizing cellulase and alginate lyase. As a carbon-based material, the biochar carrier has high biocompatibility and environmental friendliness. In previous researches, most of residual biomass after enzymolysis is directly treated, so that resource waste is caused, and only a small amount of the residual biomass is used for fertilizer development or derivative synthesis. According to the method, waste is turned into wealth, the cyclic utilization attribute is given to the waste biomass immobilization carrier after enzymolysis, the enzyme use cost is reduced, the high-value conversion rate of the gulfweed is increased, full cyclic utilization and high-value conversion of the gulfweed biomass are achieved, and a new thought is expected to be provided for development and utilization of renewable energy sources.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Sargassum biomass cellulose resource utilization, and relates to a method for constructing a double-enzyme immobilization full-cycle system by using Sargassum. BACKGROUND

[0002] Biomass energy is an important clean energy, which has experienced three generations. The first generation of biofuels is mainly edible crops, the second generation of biofuels is mainly agricultural and forestry waste, and the third generation of biofuels is mainly marine algae. It has the advantages of environmental friendliness, green sustainability and the like. Among them, Sargassum is attracting attention due to its fast growth, strong environmental adaptability and rich polysaccharides. Sargassum can produce bioethanol, bio-oil and other biofuels and other high-value products through different conversion methods. The preparation of bioethanol needs to go through pretreatment, hydrolysis and saccharification, fermentation and other steps. The common hydrolysis and saccharification methods are acid hydrolysis and enzyme hydrolysis. Acid hydrolysis usually needs to be carried out under harsh conditions such as high temperature, high pressure and strong acid. This may cause side reactions such as carbonization and dehydration of substrates and products, reducing the yield of target products. Moreover, strong acid has strong corrosion to equipment, increasing the cost of equipment maintenance and replacement. Therefore, enzyme hydrolysis is currently the most commonly used hydrolysis method, but the production cost of enzymes is relatively high, and the amount of enzymes used in the hydrolysis process is also limited, and the activity of enzymes is easily affected by temperature, pH, inhibitors and other factors.

[0003] At present, researchers mainly solve these two problems by immobilizing enzymes. Enzyme immobilization technology (physical adsorption, covalent binding, embedding / isolation, cross-linking, etc.) can simultaneously achieve the enhancement of enzyme stability and recycling by fixing enzymes on carriers (silicon-based materials, carbon-based materials, metal materials, polymer materials, composite materials, etc.). However, traditional immobilized carriers such as silica gel and ion exchange resin face double development bottlenecks. On the one hand, the raw materials are not renewable and the preparation cost is high, and on the other hand, the low-value disposal of waste carriers can easily cause secondary pollution. This kind of unsustainability is particularly prominent in the large-scale application of brown algae biorefining. SUMMARY

[0004] The purpose of the present application is to solve the problems of low utilization rate of Sargassum and high cost of enzymatic hydrolysis in the prior art, and to provide a method for constructing a double-enzyme immobilization full-cycle system by using Sargassum.

[0005] To achieve the above purpose, the following technical solutions are adopted:

[0006] The method for constructing a double-enzyme immobilization full-cycle system by using Sargassum proposed by the present application comprises the following steps:

[0007] The Sargassum raw material is put into a buffer solution, a catalyst is added for reaction, and then solid-liquid separation is carried out.

[0008] The reaction residue is fired into biochar under a nitrogen atmosphere and washed to neutral;

[0009] The neutral biochar is modified by carboxylation and activated treatment, and the catalyst is fixed on the modified biochar;

[0010] The Sargassum thunbergii raw material is added to the buffer solution and then placed in the biochar with the fixed catalyst for multi-batch reaction to realize the global utilization of Sargassum thunbergii.

[0011] Preferably, the method for obtaining the Sargassum thunbergii raw material is as follows:

[0012] The fresh Sargassum thunbergii is dried to constant weight, ground, sieved, mixed with water, and then a pretreatment reagent potassium permanganate is added. After obtaining the dispersion solution, ultrasonic and oscillation treatment are performed.

[0013] After the reaction is completed, the filter residue is obtained by centrifugal filtration, and the filter residue is washed to neutral and then dried to obtain the pretreated Sargassum thunbergii raw material.

[0014] Preferably, the mass ratio of the raw material to deionized water is 1:10-1:50, the concentration of potassium permanganate is in the range of 0.1wt%-1wt%, the ultrasonic treatment time is 20-30min, the oscillation temperature is 20-60℃, and the oscillation time is 1-8h.

[0015] Preferably, the Sargassum thunbergii raw material is placed in the buffer solution, and specifically:

[0016] The buffer solution is a citric acid-sodium citrate buffer solution, and the molar concentration of the buffer solution is in the range of 10mM / L-100mM / L, and the pH value is 4-7.

[0017] Preferably, after the Sargassum thunbergii raw material is placed in the buffer solution and the catalyst is added for reaction, solid-liquid separation is performed, and specifically:

[0018] The catalyst includes cellulase and alginate lyase;

[0019] The ratio of the buffer solution, cellulase and alginate lyase is (20mL-50mL):(0.5mL-1mL):(0.5mL-1mL), and the enzyme hydrolysis is performed at 40℃-55℃ for 24h-48h, followed by centrifugal washing to realize solid-liquid separation.

[0020] Preferably, the neutral biochar is modified by carboxylation and activated treatment, and specifically:

[0021] Step 1: After citric acid is added to ionized water and uniformly dissolved, dry biochar is added, oscillation, centrifugal filtration, and washing of the biochar is performed until the washing liquid is neutral to obtain biochar modified by surface carboxylation;

[0022] Step 2, the carboxyl-modified biochar is added into deionized water, EDC is first added and then NHS is added, and then the biochar is filtered and washed to obtain the biochar with activated surface carboxyl.

[0023] Preferably, in step 1, the ratio of deionized water to dry biochar is (50-200 mL) :(0.5-2 g); the concentration of citric acid is 0.5-3 M / L; and the oscillation is carried out at 5-35 DEG C for 6-48 h.

[0024] In step 2, the ratio of carboxyl-modified biochar to deionized water is (0.5-1 g) :(5-10 mL); the mass concentration of EDC and NHS is (1 mg / mL-10 mg / mL) :(5 mg / mL-20 mg / mL), and the standing time is 1-8 h.

[0025] Preferably, the catalyst is fixed on the modified biochar, and specifically:

[0026] The catalyst comprises cellulase and alginate lyase;

[0027] Two portions of the activated biochar are added into buffer solution, and then crude cellulase solution and crude alginate lyase solution are added respectively to realize the immobilization of the double enzymes by oscillation.

[0028] Preferably, the ratio of the activated biochar, buffer solution, crude cellulase solution and crude alginate lyase is (0.05 g-0.25 g) :(4 mL-10 mL) :(1 mL-5 mL) :(1 mL-5 mL).

[0029] Preferably, the two portions of the activated biochar are both added into buffer solution with pH of 4-8; and the double enzymes are immobilized by oscillation at 5-45 DEG C and 15-45 DEG C for 2-24 h.

[0030] Compared with the prior art, the method has the following beneficial effects:

[0031] The method for constructing a double-enzyme immobilization full-cycle system by using sargassum, the cell wall of the sargassum is mainly composed of alginate (Ca 2+ The crosslinked "egg box" structure is the core), cellulose microfibril and fucoidan, the linear polyanion chain formed by beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G) units through 1, 4-glycosidic bond, and Ca 2+Form a three-dimensional network cross-linked structure; and cellulose microfibrils and fucoidan are interpenetrated in the network through hydrogen bonds and ionic bonds to form a physical barrier against enzymatic hydrolysis, so that single cellulase may be difficult to completely decompose its cell structure. Therefore, the present application uses double enzyme co-hydrolysis, alginate lyase degrades Ca 2+ The cross-linked alginate network improves porosity, exposes the shielded cellulose, and then cellulase efficiently acts on the beta-1, 4-glucan chain, improving the enzymatic hydrolysis yield. In addition, the biochar carrier is a carbon-based material with high biocompatibility and environmental friendliness. In previous studies, only a small amount of residual biomass after enzymatic hydrolysis was used for the development of fertilizers or the synthesis of other derivatives, and most of the final residual biomass was directly treated, resulting in resource waste. Therefore, the enzymatic residue is burned into a biochar carrier for enzyme immobilization, which is used for the immobilization of cellulase and alginate lyase double enzymes, turning waste into treasure, while giving the recycling property of the immobilized carrier of the discarded biomass after enzymatic hydrolysis, reducing the use cost of the enzyme, improving the high-value conversion rate of sargassum, and realizing the full recycling and high-value conversion of sargassum biomass. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0033] Figure 1 is a schematic diagram of the method for constructing a double enzyme immobilization full cycle system by the present application using sargassum;

[0034] Figure 2 is a SEM image (a) after enzymatic hydrolysis; b) biochar after burning; c) biochar after carboxyl modification; d) biochar immobilized cellulase; e) biochar immobilized alginate lyase) of the present application;

[0035] Figure 3 is the FTIR image of the biochar before and after burning and the immobilized enzyme of the present application. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the following will combine the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.

[0037] The following detailed description of embodiments of the application provided in the accompanying drawings is not intended to limit the scope of the application as claimed, but merely represents selected embodiments of the application. Based upon the embodiments in the application, all other embodiments that a person of ordinary skill in the art obtains without creative work, fall within the scope of protection of the application.

[0038] It should be noted that similar reference numbers and letters represent similar items in the following drawings, and therefore, once an item is defined in one drawing, it need not be further defined and explained in subsequent drawings.

[0039] In the description of the embodiments of the application, it should be noted that if the terms "upper", "lower", "horizontal", "inner", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship when the product of the application is usually placed, and are only for the convenience of describing the application and simplifying the description, and therefore cannot be understood as indicating or implying that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the application. In addition, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0040] The application will be further described in detail below in conjunction with the drawings:

[0041] The application proposes a method for constructing a double-enzyme immobilized full-cycle system using sargassum, comprising the following steps:

[0042] Step 1, dry fresh sargassum to constant weight, mix with water after grinding and sieving to 80 mesh, add pre-treatment reagent potassium permanganate, obtain a dispersion solution, and then perform ultrasonic and oscillation treatment;

[0043] Step 2, after the reaction is completed, centrifugal filtration is performed to obtain filter residue, which is washed to neutral and then dried to obtain pretreated sargassum raw material;

[0044] Step 3, place the sargassum raw material into a buffer solution, then add cellulase and alginate lyase for enzymolysis, and then perform centrifugal filtration to separate solid and liquid;

[0045] Step 4, use a tube furnace to burn the enzymolysis residue into biochar under N2 atmosphere, and then wash to neutral;

[0046] Step 5, use citric acid to perform carboxyl modification on the neutral biochar, and use EDC / NHS to activate, then fix cellulase and alginate lyase on the modified biochar;

[0047] Step 6, the double enzyme immobilized on the biochar is placed in the buffer solution of step 3 again to replace the free double enzyme. After the reaction is completed, the pretreated sargassum thunbergii raw material can be added again for multi-batch enzymolysis, so as to realize the global utilization of sargassum thunbergii and improve the utilization rate of the enzyme.

[0048] As Figure 1 The flow chart of the method for constructing a double enzyme immobilization full cycle system by using sargassum thunbergii is shown in the figure, and the method is described in detail as follows:

[0049] Step 1, fresh sargassum thunbergii is repeatedly washed, dried, ground and sieved to 60-100 meshes as a raw material. The raw material is mixed with deionized water in a conical flask according to the mass ratio of 1:10-1:50, and potassium permanganate reagent (0.1-1wt%) is added. After ultrasonic treatment under the fixed conditions (10-40KHz, 60-400W, 20-60℃) for 20-30min, the pretreated raw material is placed in a constant temperature shaker (20-60℃, 1-8h) for reaction. The pretreated raw material is washed and dried to obtain a sargassum thunbergii pretreated biomass raw material.

[0050] Step 2, the pretreated raw material is poured into 10mM / L-100mM / L, pH 4-7, 20mL-50mL citric acid-sodium citrate buffer is added, and then 0.5-1mL cellulase crude enzyme solution (10mg / mL) and 0.5-1mL alginate lyase crude enzyme solution (10mg / mL) are added respectively. After enzymolysis at 40-55℃ for 24-48h, the residual biomass is centrifuged and washed to complete the first enzymolysis.

[0051] Step 3, the residual biomass of sargassum thunbergii after enzymolysis is washed and dried, and then placed in a porcelain boat and placed in a tube furnace under a nitrogen atmosphere. The initial temperature is set to 25-35℃, the temperature is raised to 550-650℃ at a rate of 5-10℃ / min and maintained for 120-180min to prepare biochar. After cooling to room temperature, the biochar is washed with warm water until it is neutral, dried in an oven and sealed for storage.

[0052] Step 4, 50-200mL of deionized water is added to a conical flask, 0.5-3M / L of citric acid is added and mixed to dissolve, and then 0.5-2g of dried biochar is added. The conical flask is placed in a constant temperature shaking box and shaken at 5-35℃ for 6-48h. After centrifugal filtration, the biochar is washed until the washing liquid is neutral, and the surface carboxyl modified biochar is obtained. The citric acid aqueous solution for modifying the biochar can be repeatedly used for multiple times.

[0053] Step 5, take 0.5-1 g of carboxyl-modified biochar and add it to 5-10 mL of deionized water. First add 1-10 mg / mL of EDC and let it stand at room temperature for 10-30 min, then add 5-20 mg / mL of NHS and let it stand for 1-8 h, then filter and wash the biochar to obtain the surface carboxyl-activated biochar.

[0054] Step 6, take two portions of 0.05-0.25 g of activated biochar and add them to 4-10 mL of PBS buffer (0.1-0.5 M) at pH 4-8, then add 1-5 mL of cellulase crude enzyme solution (10 mg / mL) and 1-5 mL of alginate lyase crude enzyme solution (10 mg / mL) respectively, and shake at 5-45°C and 15-45°C respectively for 2-24 h to immobilize the double enzymes.

[0055] Step 7, take 1-2 g of pre-treated Sargassum and add it to the citric acid-sodium citrate buffer solution in step 2, then place it in the fixed cellulase and alginate lyase in step 6 and enzymatically digest at 35-55°C for 48-72 h, then detect the glucose and alginate content in the supernatant. After the reaction is completed, add 1-2 g of pre-treated Sargassum again to continue the multi-batch enzymatic digestion.

[0056] Example 1:

[0057] A method for constructing a double enzyme immobilized full cycle system using Sargassum, comprising the following steps:

[0058] (1) Dry fresh Sargassum to constant weight, grind and sieve through an 80 mesh sieve, then mix with water and add pre-treatment reagent potassium permanganate (0.4 wt%), obtain a dispersion solution, then ultrasonic (40 KHz, 240 W, 30°C, 30 min) and oscillation treatment (40°C, 4 h);

[0059] (2) Pour the Sargassum raw material prepared in step (1) into 50 mM / L, pH 5, add 50 mL of citric acid-sodium citrate buffer, then add 1 mL of prepared cellulase crude enzyme solution (10 mg / mL) and 1 mL of alginate lyase crude enzyme solution (10 mg / mL) respectively, and enzymatically digest at 45°C for 48 h, then centrifuge and wash the residual biomass to complete the first enzymatic digestion.

[0060] (3) Take the residual biomass of the enzymatically digested Sargassum in step (2), wash and dry, then place the residual biomass in a porcelain boat and place it in a tube furnace. Set the initial temperature to 25°C under nitrogen atmosphere, increase the temperature to 550°C at a rate of 5°C / min and maintain for 120 min to prepare biochar. After cooling to room temperature, take it out and wash it with warm water until it is neutral, then place it in an oven to dry and seal for storage.

[0061] (4) 100 mL of deionized water was added to the conical flask, 2 M / L of citric acid was added and mixed to dissolve, then 1 g of the dried biochar prepared in step (3) was added. The conical flask was placed in a constant temperature shaking incubator and shaken at 25°C for 24 h. After different time, it was taken out. After centrifugal filtration, the biochar was washed until the pH of the washing liquid was 7.0, to obtain the surface carboxyl modified biochar. The citric acid aqueous solution for modifying the biochar can be repeatedly used for many times.

[0062] (5) 1 g of the carboxyl modified biochar in step (4) was added to 5 mL of deionized water. First, 5 mg / mL of EDC was added and left at room temperature for 30 min, then 10 mg / mL of NHS was added and left for 4 h, and then the biochar was filtered and washed, to obtain the surface carboxyl activated biochar.

[0063] (6) 0.2 g of the activated biochar in step (5) was taken and added to 4 mL of PBS buffer (0.1 M) with pH 5 and 7 respectively, then 1 mL of cellulase crude enzyme liquid (10 mg / mL) and 1 mL of alginate lyase crude enzyme liquid (10 mg / mL) were added respectively, and the double enzymes were immobilized by shaking at 35°C and 25°C for 12 h respectively.

[0064] (7) 1 g of the pre-treated sargassum thunbergii in step (1) was added to the citric acid-sodium citrate buffer in step (2), and then the cellulase and alginate lyase immobilized in step (6) were added, and the enzyme hydrolysis was carried out at 45°C for 72 h, and then the content of glucose and alginate in the supernatant was detected. After the reaction was completed, 1 g of the pre-treated sargassum thunbergii was added again to complete multiple batches of enzyme hydrolysis, a total of 5 times.

[0065] Example 2:

[0066] A method for constructing a double enzyme immobilized full cycle system by using sargassum thunbergii, comprising the following steps:

[0067] (1) Fresh sargassum thunbergii was dried to constant weight, ground and sieved to 60 mesh, then mixed with water, added with a pretreatment reagent potassium permanganate (0.4 wt%), and obtained a dispersion solution, then ultrasonic treatment (10 KHz, 400 W, 60°C, 20 min) and oscillation treatment (20°C, 8 h) were carried out.

[0068] (2) The sargassum thunbergii raw material prepared in step (1) was poured into 10 mM / L, pH 4, 20 mL of citric acid-sodium citrate buffer was added, and then 0.5 mL of cellulase crude enzyme liquid (10 mg / mL) and 0.5 mL of alginate lyase crude enzyme liquid (10 mg / mL) prepared respectively were added, and the enzyme hydrolysis was carried out at 45°C for 24 h, then the residual biomass was centrifuged and washed to complete the first enzyme hydrolysis.

[0069] (3) Take the residual biomass of Sargassum thunbergii in step (2) for enzymatic hydrolysis, wash and dry, and then place the residual biomass in a porcelain boat in a tube furnace. Set the initial temperature to 30°C under a nitrogen atmosphere, and increase the temperature to 600°C at a rate of 10°C / min and maintain for 150 min to prepare biochar. After cooling to room temperature, remove and wash with warm water until neutral, place in an oven to dry, and then seal and store.

[0070] (4) Add 50 mL of deionized water to a conical flask, add 0.5 M / L of citric acid, mix and dissolve, and then add 0.5 g of dried biochar prepared in step (3). Place the conical flask in a constant temperature shaking incubator and shake at 5°C for 6 h at different times. After centrifugal filtration, wash the biochar until the pH of the washing liquid is 7.0 to obtain surface carboxyl-modified biochar. The citric acid aqueous solution for modifying biochar can be used repeatedly for multiple times.

[0071] (5) Take 0.5 g of carboxyl-modified biochar in step (4) and add to 6 mL of deionized water. First, add 1 mg / mL of EDC and stand at room temperature for 10 min, then add 5 mg / mL of NHS and stand for 4 h, and then filter and wash the biochar to obtain surface carboxyl-activated biochar.

[0072] (6) Take two activated 0.05 g of biochar in step (5) and add to 6 mL of PBS buffer (0.3 M) at pH 4 and 5, respectively, and then add 2 mL of crude cellulase solution (10 mg / mL) and 2 mL of crude alginate lyase solution (10 mg / mL), respectively. Shake at 5°C and 15°C for 2 h to immobilize the double enzymes.

[0073] (7) Take 1 g of pretreated Sargassum thunbergii in step (1) and add to the citric acid-sodium citrate buffer in step (2), and then place in the fixed cellulase and alginate lyase in step (6) at 35°C for 48 h of enzymatic hydrolysis. Detect the contents of glucose and alginate in the supernatant. After the reaction is completed, add 1 g of pretreated Sargassum thunbergii again to complete multiple batches of enzymatic hydrolysis, a total of 5 times.

[0074] Example 3:

[0075] A method for constructing a double enzyme immobilized full cycle system using Sargassum thunbergii, comprising the following steps:

[0076] (1) Dry fresh Sargassum thunbergii to constant weight, grind, pass through a 100 mesh sieve, mix with water, add a pretreatment reagent potassium permanganate (0.1 wt%), obtain a dispersion solution, and then ultrasonic (30 KHz, 320 W, 50°C, 25 min) and oscillation (30°C, 6 h) treatment;

[0077] (2) The Sargassum na tans of step (1) were poured into 100 mM / L, pH 7, 50 mL of citric acid-sodium citrate buffer, and 0.6 mL of prepared cellulase crude enzyme solution (10 mg / mL) and 0.8 mL of alginate lyase crude enzyme solution (10 mg / mL) were added, respectively. After enzymolysis at 45°C for 36 h, the residual biomass was centrifuged and washed to complete the first enzymolysis.

[0078] (3) The residual biomass of enzymolyzed Sargassum na tans of step (2) was washed and dried, and then placed in a porcelain boat in a tube furnace. The initial temperature was set at 35°C under a nitrogen atmosphere, and the temperature was raised to 650°C at a rate of 8°C / min and maintained for 180 min to prepare biochar. After cooling to room temperature, the biochar was washed to neutral with warm water, dried in an oven, and then sealed and stored.

[0079] (4) 200 mL of deionized water was added to a conical flask, 3 M / L of citric acid was added and mixed to dissolve, and then 2 g of dried biochar prepared in step (3) was added. The conical flask was placed in a constant-temperature shaking incubator and shaken at 35°C for 48 h. After shaking, the biochar was centrifuged and washed until the pH of the washing liquid was 7.0. The surface carboxyl-modified biochar was obtained, and the citric acid aqueous solution for modifying the biochar could be repeatedly used for multiple times.

[0080] (5) 0.7 g of the carboxyl-modified biochar in step (4) was added to 8 mL of deionized water. First, 10 mg / mL of EDC was added and allowed to stand at room temperature for 20 min, then 20 mg / mL of NHS was added and allowed to stand for 8 h. The biochar was filtered and washed to obtain the surface carboxyl-activated biochar.

[0081] (6) Two activated biochars of 0.25 g each in step (5) were added to 4 mL of PBS buffer (0.5 M) at pH 6 and 8, respectively. Then 5 mL of cellulase crude enzyme solution (10 mg / mL) and 5 mL of alginate lyase crude enzyme solution (10 mg / mL) were added, respectively. The double enzymes were immobilized by shaking at 25°C and 35°C for 24 h, respectively.

[0082] (7) 2 g of pretreated Sargassum na tans in step (1) was added to the citric acid-sodium citrate buffer in step (2), and then placed in the cellulase and alginate lyase immobilized in step (6) for enzymolysis at 55°C for 60 h. The contents of glucose and alginate in the supernatant were detected. After the reaction was completed, 2 g of pretreated Sargassum na tans was added again to complete multiple batches of enzymolysis, a total of 5 times.

[0083] Example 4:

[0084] A method for constructing a double-enzyme immobilized full-cycle system using Sargassum na tans, comprising the following steps:

[0085] (1) Fresh Sargassum thunbergii was dried to constant weight, ground, and sieved to 100 mesh. The sieved material was mixed with water and added with KMn04(1 wt%) as a pretreatment reagent. The resulting dispersion was treated by ultrasonication (20 KHz, 60 W, 20 °C, 30 min) and oscillation (60 °C, 1 h).

[0086] (2) The Sargassum thunbergii material prepared in step (1) was poured into 40 mL of a citric acid-sodium citrate buffer with a pH of 6.0. Then, 0.7 mL of a crude cellulase solution (10 mg / mL) and 0.9 mL of a crude alginate lyase solution (10 mg / mL) were added. The mixture was subjected to enzymatic hydrolysis at 45 °C for 24 h. The residual biomass was then washed by centrifugation to complete the first enzymatic hydrolysis.

[0087] (3) The residual biomass from step (2) was washed and dried, and then placed in a porcelain boat in a tube furnace. The initial temperature was set to 25 °C under a nitrogen atmosphere, and the temperature was increased to 600 °C at a rate of 10 °C / min and maintained for 120 min to prepare the biochar. After cooling to room temperature, the biochar was removed, washed with warm water until neutral, dried in an oven, and then sealed for storage.

[0088] (4) 150 mL of deionized water was added to a conical flask, and 1 M / L of citric acid was added and mixed to dissolve. Then, 1.5 g of the dried biochar prepared in step (3) was added. The conical flask was placed in a constant-temperature shaking incubator and shaken at 15 °C for 36 h. After different time periods, the conical flask was removed. The biochar was washed by centrifugation until the pH of the washing solution was 7.0. The surface carboxyl-modified biochar was obtained, and the citric acid aqueous solution used for modifying the biochar could be reused multiple times.

[0089] (5) 1 g of the carboxyl-modified biochar from step (4) was added to 10 mL of deionized water. First, 8 mg / mL of EDC was added and allowed to stand at room temperature for 30 min. Then, 16 mg / mL of NHS was added and allowed to stand for 6 h. The biochar was filtered and washed to obtain the surface carboxyl-activated biochar.

[0090] (6) Two portions of 0.15 g of the activated biochar from step (5) were added to 8 mL of PBS buffer (0.3 M) with a pH of 5 and 6, respectively. Then, 3 mL of a crude cellulase solution (10 mg / mL) and 3 mL of a crude alginate lyase solution (10 mg / mL) were added, respectively. The two enzymes were immobilized by shaking at 25 °C and 45 °C, respectively, for 12 h.

[0091] (7) Take 2 g of the pre-processed Sargassum thunbergii from step (1) and add it to the citric acid-sodium citrate buffer solution in step (2). Then, place it in the fixed cellulase and alginate lyase in step (6) and enzymatically hydrolyze it at 35°C for 72 hours. Then, detect the glucose and alginate content in the supernatant. After the reaction is completed, add 1 g of pre-processed Sargassum thunbergii again to continue the multi-batch enzymatic hydrolysis, a total of 5 times.

[0092] Example 5:

[0093] A method for constructing a double-enzyme immobilized full-cycle system using Sargassum thunbergii, comprising the following steps:

[0094] (1) Dry fresh Sargassum thunbergii to a constant weight, grind it, sieve it through an 80-mesh sieve, mix it with water, add pre-treatment reagent potassium permanganate (0.5 wt%), obtain a dispersion solution, and then ultrasonic (30 KHz, 120 W, 50°C, 30 min) and oscillation (40°C, 4 h) treatment.

[0095] (2) Pour the Sargassum thunbergii raw material prepared in step (1) into 40 mM / L, pH 4, 30 mL of citric acid-sodium citrate buffer solution, and then add 0.8 mL of prepared cellulase crude enzyme solution (10 mg / mL) and 1 mL of prepared alginate lyase crude enzyme solution (10 mg / mL), respectively. Enzymatically hydrolyze at 45°C for 48 h, then centrifuge and wash the residual biomass to complete the first enzymatic hydrolysis.

[0096] (3) Take the residual biomass of the enzymatically hydrolyzed Sargassum thunbergii in step (2), wash and dry it, place the residual biomass in a porcelain boat, and place it in a tube furnace. Set the initial temperature to 30°C under a nitrogen atmosphere, increase the temperature to 650°C at a rate of 5°C / min and maintain it for 150 min to prepare biochar. After cooling to room temperature, remove it, wash it with warm water until it is neutral, place it in an oven to dry, and then seal and store it.

[0097] (4) Add 50 mL of deionized water to a conical flask, add 2 M / L of citric acid, mix well to dissolve, and then add 1.5 g of dried biochar prepared in step (3). Place the conical flask in a constant-temperature shaking incubator and shake it at 15°C for 12 h. Then remove it. Centrifuge and wash the biochar until the washing liquid pH is 7.0 to obtain surface carboxylated modified biochar. The citric acid aqueous solution used to modify the biochar can be repeatedly used multiple times.

[0098] (5) Take 0.9 g of the carboxylated modified biochar in step (4) and add it to 5 mL of deionized water. First, add 7 mg / mL of EDC and let it stand at room temperature for 20 min, then add 15 mg / mL of NHS and let it stand for 8 h. Then filter and wash the biochar to obtain surface carboxylated activated biochar.

[0099] (6) Take two 0.15 g of activated biochar from step (5) and add them to 4 mL of PBS buffer (0.5 M) at pH 4 and 7, respectively. Then add 2 mL of cellulase crude enzyme solution (10 mg / mL) and 3 mL of alginate lyase crude enzyme solution (10 mg / mL), respectively. Shake at 5°C and 15°C for 12 h to immobilize the double enzymes.

[0100] (7) Take 2 g of pre-treated Sargassum thunbergii from step (1) and add it to the citric acid-sodium citrate buffer solution in step (2). Then add the immobilized cellulase and alginate lyase from step (6) and incubate at 55°C for 48 h. Measure the glucose and alginate content in the supernatant. After the reaction is completed, add 1 g of pre-treated Sargassum thunbergii again to complete multiple batches of enzyme hydrolysis, a total of 5 times.

[0101] Example 6:

[0102] A method for constructing a double-enzyme immobilized full-cycle system using Sargassum thunbergii, comprising the following steps:

[0103] (1) Dry fresh Sargassum thunbergii to constant weight, grind and sieve to 60 mesh, then mix with water, add pre-treatment reagent potassium permanganate (0.8 wt%), obtain a dispersion solution, and then ultrasonic (40 KHz, 60 W, 40°C, 20 min) and oscillation treatment (50°C, 6 h).

[0104] (2) Pour the Sargassum thunbergii raw material prepared in step (1) into 50 mM / L, pH 4, add 50 mL of citric acid-sodium citrate buffer solution, then add 0.8 mL of cellulase crude enzyme solution (10 mg / mL) and 0.7 mL of alginate lyase crude enzyme solution (10 mg / mL) respectively, and incubate at 45°C for 24 h. After centrifugation and washing of the residual biomass, the first enzyme hydrolysis is completed.

[0105] (3) Take the residual biomass of Sargassum thunbergii after enzyme hydrolysis in step (2), wash and dry, then place the residual biomass in a porcelain boat and place it in a tube furnace. Set the initial temperature to 25°C under nitrogen atmosphere, increase the temperature to 550°C at a rate of 10°C / min and maintain for 180 min to prepare biochar. After cooling to room temperature, remove and wash with warm water until neutral, then dry in an oven and seal for storage.

[0106] (4) Add 50 mL of deionized water to a conical flask, add 2.5 M / L of citric acid and mix to dissolve, then add 1.7 g of dry biochar prepared in step (3). Place the conical flask in a constant temperature shaking incubator and shake at 15°C for 36 h. After taking out, centrifuge and wash the biochar until the pH of the washing solution is 7.0. The surface carboxylated modified biochar is obtained, and the citric acid aqueous solution for modifying the biochar can be used repeatedly.

[0107] (5) Take 1 g of carboxyl-modified biochar in step (4) and add it to 10 mL of deionized water. First, add 6 mg / mL of EDC and stand at room temperature for 10 min, then add 15 mg / mL of NHS and stand for 8 h, and then filter and wash the biochar to obtain the surface carboxyl-activated biochar.

[0108] (6) Take 0.25 g of activated biochar in step (5) and add it to 10 mL of PBS buffer (0.2 M) with pH 4 and 5, respectively, and then add 3 mL of cellulase crude enzyme solution (10 mg / mL) and 2 mL of alginate lyase crude enzyme solution (10 mg / mL), respectively. The double enzymes are immobilized by oscillation at 35°C and 35°C for 24 h, respectively.

[0109] (7) Take 1 g of pre-treated Sargassum thunbergii in step (1) and add it to the citric acid-sodium citrate buffer in step (2), and then place it in the cellulase and alginate lyase immobilized in step (6) for enzymatic hydrolysis at 45°C for 60 h. The glucose and alginate contents in the supernatant are detected. After the reaction is completed, 2 g of pre-treated Sargassum thunbergii is added again to complete multiple batches of enzymatic hydrolysis, a total of 5 times.

[0110] Example 7:

[0111] A method for constructing a double-enzyme immobilized full-cycle system using Sargassum thunbergii, comprising the following steps:

[0112] (1) Dry fresh Sargassum thunbergii to constant weight, grind and sieve to 80 mesh, mix with water, add pre-treatment reagent potassium permanganate (0.4 wt%), obtain a dispersion solution, and then ultrasonic (40 KHz, 320 W, 20°C, 25 min) and oscillation (30°C, 6 h) treatment;

[0113] (2) Pour the Sargassum thunbergii raw material prepared in step (1) into 100 mM / L, pH 4, add 40 mL of citric acid-sodium citrate buffer, and then add 1 mL of cellulase crude enzyme solution (10 mg / mL) and 0.9 mL of alginate lyase crude enzyme solution (10 mg / mL) prepared respectively, and centrifuge and wash the residual biomass after enzymatic hydrolysis at 45°C for 36 h to complete the first enzymatic hydrolysis.

[0114] (3) Take the residual biomass of enzymatically hydrolyzed Sargassum thunbergii in step (2), wash and dry, place the residual biomass in a porcelain boat, and place it in a tube furnace. Set the initial temperature to 25°C under nitrogen atmosphere, increase the temperature to 550°C at a rate of 5°C / min and maintain for 120 min to prepare biochar. After cooling to room temperature, wash with warm water to neutral, dry in an oven, and seal for storage.

[0115] (4) Take 200 mL of deionized water into a conical flask, add 0.5 M / L of citric acid, mix and dissolve, then take 1 g of the dried biochar prepared in step (3) and add it into the conical flask. Place the conical flask in a constant temperature shaking incubator and shake at 5°C for 48 hours. After different time, take it out. After centrifugal filtration, wash the biochar until the pH of the washing liquid is 7.0 to obtain the surface carboxylated modified biochar. The citric acid aqueous solution for modifying the biochar can be repeatedly used for multiple times.

[0116] (5) Take 0.8 g of the carboxylated modified biochar in step (4) and add it into 10 mL of deionized water. First, add 10 mg / mL of EDC and stand at room temperature for 10 min, then add 5 mg / mL of NHS and stand for 8 h, and then filter and wash the biochar to obtain the surface carboxylated activated biochar.

[0117] (6) Take 0.05 g of the activated biochar in step (5) and add it into 7 mL of PBS buffer (0.1 M) with pH of 8 and 4, respectively, then add 1 mL of cellulase crude enzyme liquid (10 mg / mL) and 1 mL of alginate lyase crude enzyme liquid (10 mg / mL), respectively, and shake at 45°C and 35°C, respectively, for 12 h to immobilize the double enzymes.

[0118] (7) Take 2 g of the pre-processed sargassum in step (1) and add it into the citric acid-sodium citrate buffer in step (2), and then place it into the cellulase and alginate lyase immobilized in step (6) for enzymolysis at 55°C for 60 h, and then detect the contents of glucose and alginate in the supernatant. After the reaction is completed, add 1 g of the pre-processed sargassum again to continue the multi-batch enzymolysis, and a total of 5 times.

[0119] Example 8:

[0120] A method for constructing a double enzyme immobilized full cycle system by using sargassum, comprising the following steps:

[0121] (1) Dry the fresh sargassum to a constant weight, grind and sieve through a 60 mesh sieve, then mix with water, add a pre-treatment reagent potassium permanganate (0.6 wt%), obtain a dispersion solution, and then perform ultrasonic treatment (30 KHz, 400 W, 30°C, 30 min) and oscillation treatment (40°C, 4 h);

[0122] (2) Pour the sargassum raw material prepared in step (1) into 20 mL of citric acid-sodium citrate buffer with pH of 7, then add 0.9 mL of cellulase crude enzyme liquid (10 mg / mL) and 0.6 mL of alginate lyase crude enzyme liquid (10 mg / mL) configured respectively, and perform enzymolysis at 45°C for 24 h, then centrifugal wash the residual biomass to complete the first enzymolysis.

[0123] (3) Take the residual biomass of Sargassum thunbergii in step (2) for enzymatic hydrolysis, wash and dry, and then place the residual biomass in a porcelain boat in a tube furnace. Set the initial temperature to 25°C under a nitrogen atmosphere, and increase the temperature to 600°C at a rate of 5°C / min and maintain for 120 min to prepare biochar. After cooling to room temperature, remove and wash with warm water until neutral, place in an oven to dry, and then seal and store.

[0124] (4) Add 200 mL of deionized water to a conical flask, add 0.5 M / L of citric acid, mix and dissolve, and then add 0.5 g of dried biochar prepared in step (3). Place the conical flask in a constant temperature shaking incubator and shake at 35°C for 6 h at different times. After centrifugal filtration, wash the biochar until the pH of the washing liquid is 7.0 to obtain surface carboxyl-modified biochar. The citric acid aqueous solution for modifying biochar can be used repeatedly for multiple times.

[0125] (5) Take 0.7 g of carboxyl-modified biochar in step (4) and add to 8 mL of deionized water. First add 10 mg / mL of EDC and stand at room temperature for 20 min, then add 5 mg / mL of NHS and stand for 8 h, and then filter and wash the biochar to obtain surface carboxyl-activated biochar.

[0126] (6) Take two portions of 0.25 g of activated biochar in step (5) and add to 4 mL of PBS buffer (0.5 M) at pH 8 and 4, respectively. Then add 1 mL of crude cellulase solution (10 mg / mL) and 4 mL of crude alginate lyase solution (10 mg / mL), respectively. Shake at 45°C and 25°C, respectively, for 24 h to immobilize the double enzymes.

[0127] (7) Take 1 g of pretreated Sargassum thunbergii in step (1) and add to the citric acid-sodium citrate buffer in step (2). Then place in the fixed cellulase and alginate lyase in step (6) and hydrolyze at 45°C for 60 h. Detect the contents of glucose and alginate in the supernatant. After the reaction is completed, add 1 g of pretreated Sargassum thunbergii again to complete multiple batches of enzyme hydrolysis, a total of 5 times.

[0128] Example 9:

[0129] A method for constructing a double enzyme immobilized full cycle system using Sargassum thunbergii, comprising the following steps:

[0130] (1) Dry fresh Sargassum thunbergii to constant weight, grind, sieve through a 60 mesh sieve, mix with water, add a pretreatment reagent potassium permanganate (0.5 wt%), obtain a dispersion solution, and then ultrasonic (20 KHz, 240 W, 60°C, 20 min) and oscillation (30°C, 2 h) treatment;

[0131] (2) Pour the Sargassum raw material obtained in step (1) into a 100mM / L buffer solution with a pH of 4 and add 50mL of citrate-sodium citrate buffer solution. Then add 0.9mL of prepared crude cellulase solution (10mg / mL) and 0.8mL of crude alginate lyase solution (10mg / mL). After enzymatic hydrolysis at 45℃ for 36h, centrifuge and wash the residual biomass to complete the first enzymatic hydrolysis.

[0132] (3) Take the residual biomass from the enzymatic hydrolysis of Sargassum in step (2), wash and dry it, then place the residual biomass in a ceramic boat and put it in a tube furnace. Set the initial temperature to 25℃ under a nitrogen atmosphere, increase the temperature to 550℃ at 10℃ / min and hold for 120min to prepare biochar. After cooling to room temperature, take it out, wash it with warm water until neutral, put it in an oven to dry, and then seal and store it.

[0133] (4) Add 150 mL of deionized water to an Erlenmeyer flask, add 3 M / L citric acid, mix well and dissolve, then add 2 g of the dried biochar prepared in step (3). Place the Erlenmeyer flask in a constant temperature shaking oven and shake at 5 °C for 48 h for different times, then remove it. After centrifugation and filtration, wash the biochar until the pH of the washing solution is 7.0 to obtain surface carboxylated modified biochar. The citric acid aqueous solution used for modifying the biochar can be reused multiple times.

[0134] (5) Take 0.5g of the carboxylated biochar from step (4) and add it to 10mL of deionized water. First, add 1mg / mL of EDC and let it stand at room temperature for 30min. Then add 20mg / mL of NHS and let it stand for another 1h. Filter and wash the biochar to obtain biochar with activated carboxyl groups on the surface.

[0135] (6) Take two portions of activated 0.25g biochar from step (5) and add them to 6mL of PBS buffer (0.5M) at pH 4 and 8 respectively. Then add 1mL of crude cellulase solution (10mg / mL) and 1mL of crude alginate lyase solution (10mg / mL) respectively. Shake at 45℃ and 15℃ for 2h respectively to immobilize the two enzymes.

[0136] (7) Take 2g of the pretreated Sargassum from step (1) and add it to the citrate-sodium citrate buffer solution from step (2). Then, add the cellulase and alginate lyase fixed in step (6) and perform enzymatic hydrolysis at 40℃ for 48h. Detect the glucose and alginate content in the supernatant. After the reaction is complete, add another 2g of pretreated Sargassum to continue multiple batches of enzymatic hydrolysis, for a total of 5 times.

[0137] Example 10:

[0138] A method for constructing a dual-enzyme immobilized full-cycle system using Sargassum fusiforme includes the following steps:

[0139] (1) Fresh Sargassum thunbergii was dried to constant weight, ground, and sieved to 60 mesh. The sieved material was mixed with water and 0.3 wt% KMnO4 was added. The mixture was ultrasonicated (30 KHz, 320 W, 50°C, 25 min) and shaken (30°C, 4 h).

[0140] (2) The Sargassum thunbergii obtained in step (1) was poured into 80 mM / L, pH 7, 40 mL citric acid-sodium citrate buffer, and 0.8 mL of cellulase crude enzyme solution (10 mg / mL) and 0.5 mL of alginate lyase crude enzyme solution (10 mg / mL) were added. The mixture was enzymatically hydrolyzed at 45°C for 48 h, and the residual biomass was washed by centrifugation to complete the first enzymatic hydrolysis.

[0141] (3) The residual biomass obtained in step (2) was washed and dried, and then placed in a porcelain boat in a tube furnace. The initial temperature was set to 35°C, and the temperature was increased to 550°C at a rate of 5°C / min and maintained for 120 min to prepare biochar under a nitrogen atmosphere. After cooling to room temperature, the biochar was washed with warm water until it was neutral, dried in an oven, and then sealed and stored.

[0142] (4) 100 mL of deionized water was added to a conical flask, 1 M / L citric acid was added and mixed to dissolve, and then 1.5 g of dried biochar obtained in step (3) was added. The conical flask was placed in a constant temperature shaking incubator and shaken at 25°C for 36 h. After shaking, the biochar was centrifuged and washed until the pH of the washing solution was 7.0. The surface carboxylated modified biochar was obtained, and the citric acid aqueous solution used for modification of the biochar could be reused multiple times.

[0143] (5) 0.8 g of the carboxylated modified biochar obtained in step (4) was added to 5 mL of deionized water. 9 mg / mL of EDC was added and allowed to stand at room temperature for 30 min, then 15 mg / mL of NHS was added and allowed to stand for 8 h. The biochar was filtered and washed to obtain surface carboxylated activated biochar.

[0144] (6) Two 0.2 g portions of the activated biochar obtained in step (5) were added to 4 mL of PBS buffer (0.3 M) at pH 6 and 8, respectively. Then, 5 mL of cellulase crude enzyme solution (10 mg / mL) and 1 mL of alginate lyase crude enzyme solution (10 mg / mL) were added, respectively. The two enzymes were immobilized by shaking at 25°C and 35°C, respectively, for 12 h.

[0145] (7) Take 1.5 g of Sargassum after pretreatment in step (1) and add it to the citric acid-sodium citrate buffer in step (2), and then place it in the fixed cellulase and alginate lyase in step (6) for enzymatic hydrolysis at 55°C for 60 h. After the reaction is completed, 1.5 g of pretreated Sargassum is added again to continue the multi-batch enzymolysis, and a total of 5 times.

[0146] The products obtained in the above 10 groups of experiments were analyzed to obtain:

[0147] The glucose content of the supernatant of the enzymolysis products of the 10 groups of experiments was measured using a biosensor, and the alginate content was determined according to HG / T5932-2021 "Fertilizer efficiency agent-alginate" intermediate hydroxy biphenyl spectrophotometry.

[0148] Table 1 Test results of sample components under different enzymolysis conditions

[0149]

[0150] The advantages of immobilized enzymes are that they can not only significantly enhance the conformational stability of enzyme molecules, but also improve the reusability of enzymes, and realize low-cost multi-cycle catalysis. As can be seen from Table 1, the immobilized double enzymes can maintain a yield of more than 50% in the continuous 5 rounds of enzymolysis reaction.

[0151] Figure 2 It is shown that the surface of Sargassum biomass after enzymolysis is dense and rough, and when the biochar is fired, the volatile matter escapes to form pores, the carbon skeleton is restructured, and the surface presents a honeycomb or network multi-level pore structure with increased specific surface area. The citric acid carboxyl modification increases the number of biochar pores, and part of the impurities and fragments are washed out by acidification. After immobilization of the enzyme, the surface of the biochar is covered with granular or film-shaped enzyme molecules, and part of the pores are filled, but the main pore channels are retained, indicating that the enzyme does not completely block the pore channels.

[0152] Figure 3 It is shown that the enzymolysis biomass has a strong absorption peak at 3400 cm -1 due to the presence of hydroxyl groups (-OH) in polysaccharides, and when the biochar is fired, the polysaccharides are decomposed by high-temperature pyrolysis, part of the hydroxyl groups are dehydrated to form aromatic rings and other structures, resulting in a decrease in the intensity of the -OH peak. After immobilization of the enzyme, the -NH stretching vibration peak at 3300 cm -1 is significantly enhanced due to the condensation of the amino group (-NH2) in the enzyme with the carboxyl group (-COOH) in the biochar to form an amide bond (-CONH-). Oxidation introduces a carboxyl group (-COOH), which produces a characteristic peak near 1600 cm -1 , which is superimposed with the aromatic C=C skeleton vibration formed by high-temperature carbonization of the biochar in this region. The 1550-1510 cm -1The absorption peak at 1300-1200 cm corresponds to the amide II band -1 The absorption peak at 876 cm corresponds to the C-N stretching vibration and N-H in-plane bending vibration, both of which are characteristic absorption regions of amide bonds, confirming that the enzyme is immobilized on the surface of the biochar. -1 The absorption peak at 810 cm corresponds to the out-of-plane bending vibration of C-H on the aromatic ring, indicating that a highly graphitized aromatic structure is formed during the calcination process. This peak still exists after the immobilization of the enzyme, indicating that the enzyme binding does not significantly damage the aromatic skeleton of the biochar, and the carrier structure has good stability.

[0153] The above is only a preferred embodiment of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a double-enzyme immobilized whole-cycle system using Sargassum, characterized by, The method comprises the following steps: The sargassum material is placed in a buffer solution, a catalyst is added for reaction, and then solid-liquid separation is performed; The reaction residue is calcined into biochar under a nitrogen atmosphere and washed to neutral; The neutral biochar is subjected to carboxyl modification and activation treatment, and the catalyst is fixed on the modified biochar; The sargassum material is added to the buffer solution and then placed in the biochar with the fixed catalyst for multi-batch reaction, so as to realize the global utilization of sargassum.

2. The method of constructing a double-enzyme immobilized whole-cycle system using sargassum according to claim 1, characterized in that, The method for obtaining the sargassum material is as follows: Fresh sargassum is dried to constant weight, ground, sieved, mixed with water, and then a pretreatment reagent potassium permanganate is added, and the obtained dispersion solution is subjected to ultrasonic and oscillation treatment; After the reaction is completed, centrifugal filtration is performed to obtain filter residue, the filter residue is washed to neutral, and then dried to obtain the pretreated sargassum material.

3. The method of constructing a double-enzyme immobilized whole-cycle system using Sargassum according to claim 2, characterized in that, The mass ratio of the raw material to deionized water is 1:10-1:50, the concentration of potassium permanganate is 0.1wt%-1wt%, the ultrasonic treatment time is 20-30min, the oscillation temperature is 20-60℃, and the oscillation time is 1-8h.

4. The method of constructing a double-enzyme immobilized whole-cycle system using sargassum according to claim 1, characterized in that, The sargassum material is placed in a buffer solution, and the buffer solution is a citric acid-sodium citrate buffer solution, the molar concentration of the buffer solution is 10mM / L-100mM / L, and the pH value is 4-7. The sargassum material is placed in a buffer solution, a catalyst is added for reaction, and then solid-liquid separation is performed; 5. The method of constructing a double-enzyme immobilized whole-cell recycling system using Sargassum according to claim 1, characterized in that, The catalyst comprises cellulase and alginate lyase; The buffer solution, cellulase and alginate lyase are in a ratio of (20mL-50mL):(0.5mL-1mL):(0.5mL-1mL), and the enzyme hydrolysis is performed at 40℃-55℃ for 24h-48h, followed by centrifugal washing to realize solid-liquid separation. The neutral biochar is subjected to carboxyl modification and activation treatment, and the catalyst is fixed on the modified biochar; 6. The method of constructing a double-enzyme immobilized whole-cell recycling system using Sargassum according to claim 1, characterized in that, Step 1: citric acid is added to ionized water, mixed and dissolved, and then dry biochar is added, oscillation, centrifugal filtration and washing of the biochar are performed until the washing liquid is neutral, and the biochar modified by surface carboxyl is obtained; Step 2: the biochar modified by carboxyl is taken and added to deionized water, EDC is first added, and then NHS is added, and the biochar is filtered and washed to obtain biochar activated by surface carboxyl. In step 1, the ratio of deionized water to dry biochar is (50-200mL):(0.5-2g), the molar concentration of citric acid is 0.5-3M / L, and oscillation is performed at 5℃-35℃ for 6h-48h; 7. The method of constructing a double-enzyme immobilized whole-cell recycling system using Sargassum according to claim 6, characterized by, In step 2, the ratio of the biochar modified by carboxyl to deionized water is (0.5-1g):(5-10mL), the mass concentration of EDC and NHS is (1mg / mL-10mg / mL):(5mg / mL-20mg / mL), and the standing time is 1h-8h. The catalyst comprises cellulase and alginate lyase; 8. The method of constructing a double-enzyme immobilized whole-cell recycling system using Sargassum according to claim 1, characterized in that, Two portions of the activated biochar are added to a buffer solution, and then cellulase crude enzyme liquid and alginate lyase crude enzyme liquid are added respectively, and oscillation is performed to realize the immobilization of the double enzymes. ​ ​ 9. The method of constructing a double-enzyme immobilized whole-cell recycling system using Sargassum according to claim 8, characterized by, The ratio of activated biochar, buffer, cellulase crude enzyme solution and alginate lyase after activation is (0.05g-0.25g):(4mL-10mL):(1mL-5mL):(1mL-5mL).

10. The method of constructing a double-enzyme immobilized whole-cell recycling system using Sargassum according to claim 8, characterized by, Both activated biochars are added to the buffer with pH of 4-8; the double enzymes are immobilized by oscillation at 5-45℃ and 15-45℃ for 2-24h, respectively. Both activated biochars are added to the buffer with pH of 4-8; the double enzymes are immobilized by oscillation at 5-45℃ and 15-45℃ for 2-24h, respectively.

Images