CN113862320A - Method for separating protein from antibiotic fungi residues - Google Patents
Method for separating protein from antibiotic fungi residues Download PDFInfo
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- CN113862320A CN113862320A CN202110938820.5A CN202110938820A CN113862320A CN 113862320 A CN113862320 A CN 113862320A CN 202110938820 A CN202110938820 A CN 202110938820A CN 113862320 A CN113862320 A CN 113862320A
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Abstract
The invention relates to a method for separating protein from antibiotic fungi residues, which comprises the steps of hydrolyzing the antibiotic fungi residues by using magnetic biochar immobilized enzyme, and separating the protein from the antibiotic fungi residues; the magnetic biochar immobilized enzyme is a complex enzyme compounded by lysozyme and neutral protease, and the magnetic biochar is used as a carrier on which the lysozyme and the neutral protease are fixed. The invention obtains the optimal complex enzyme by screening and takes the magnetic biochar as a carrier to carry out immobilization treatment on the enzyme so as to resistThe biotoxin bacterial dregs are subjected to resource and reduction treatment, so that the antibiotic residual quantity in the bacterial dregs is reduced. The magnetic biochar is used as a carrier of the immobilized enzyme, has the characteristics of large specific surface area, porosity and surface modification, and overcomes the defect of magnetic Fe3O4Easy agglomeration of nano particles, small specific surface area and the like. The carrier takes the mushroom dregs as raw materials, the raw materials are sufficient and easy to obtain, and the mushroom dregs have the same magnetic characteristic, and can be separated and recovered by a method of an external magnetic field, so that the utilization efficiency of the enzyme is improved.
Description
Technical Field
The invention relates to the technical field of antibiotic fungi residue treatment, in particular to a method for separating protein from antibiotic fungi residue.
Background
According to statistics, the annual yield of antibiotics in China is about 24.8 ten thousand tons, and a large amount of high-water-content fermentation residues, namely antibiotic bacterial residues, are generated in the production process, and the yield of the antibiotics reaches more than 200 ten thousand t/year. The incineration cost of the mushroom dregs becomes a bottleneck for the survival and development of the antibiotic industry.
For the treatment of antibiotic dregs, a more appropriate way for disposal is always sought. Research on harmless treatment and different purposes of antibiotic bacterial residues is carried out by a plurality of scientific research institutions and pharmaceutical enterprises. The enzyme method is a method for breaking the cell wall of the microorganism by utilizing the hydrolysis of enzyme and releasing substances inside and outside the cell to achieve the purpose of protein extraction, and the enzyme method is feasible for extracting the protein in the residual antibiotic bacterial residues. The method for extracting the protein in the antibiotic residues by adopting the enzymolysis mode is beneficial to realizing high-value resource utilization of the antibiotic residues, reducing the environmental and health hazards caused by pollution of the antibiotic residues, and providing a new direction for resource treatment of the antibiotic residues and research and development of environment-friendly recycling technology. Enzymolysis can not only destroy the cell structure, but also destroy the combination of protein and other water insoluble matters. However, when the free enzyme is used for extracting protein, the consumption of the enzyme is large, the cost is high, the direct addition of the free enzyme is easily influenced by some extreme conditions, such as strong acid, strong alkali, high temperature, organic reagents and the like, the free enzyme is also very difficult to recycle, and the economic benefit is not high.
Disclosure of Invention
Technical problem to be solved
In view of the above disadvantages and shortcomings of the prior art, the present invention provides a method for separating protein from antibiotic fungi residues, which utilizes the hydrolysis of magnetic biochar immobilized enzyme to break the cell wall of microorganism and release intracellular and extracellular substances, thereby achieving the purpose of protein extraction. The technology utilizes the characteristics that the magnetic biochar immobilized enzyme has high enzyme activity, can be recycled and recycled for multiple times, still keeps the high enzyme activity, improves the utilization rate of the enzyme, and reduces the cost for separating protein from antibiotic fungi residues.
(II) technical scheme
In order to achieve the purpose, the invention adopts the main technical scheme that:
a method for separating protein from antibiotic fungi residues comprises hydrolyzing the antibiotic fungi residues by using magnetic biochar immobilized enzyme, and separating protein from the antibiotic fungi residues; the magnetic biochar immobilized enzyme is a complex enzyme compounded by lysozyme and neutral protease, and the magnetic biochar is used as a carrier on which the lysozyme and the neutral protease are fixed.
According to a preferred embodiment of the invention, the complex enzyme is prepared by compounding lysozyme and neutral protease according to the mass ratio of 1: 1-2: 1; more preferably, the complex enzyme is prepared by compounding lysozyme and neutral protease according to the mass ratio of 2: 1.
According to the preferred embodiment of the invention, the mass ratio of the complex enzyme to the magnetic biochar is 1: 2-7.
According to the preferred embodiment of the invention, the pH value of the antibiotic dreg liquid is adjusted to 7-9, magnetic biochar immobilized complex enzyme is added, the reaction temperature is kept at 50-55 ℃, a constant-temperature shaking table is used for 60-150r/min, continuous stirring and enzymolysis are carried out for 2-3h, the immobilized enzyme is separated and recovered in an external magnetic field after the reaction is finished, the enzyme is inactivated for 10-20min under the condition of water bath at 100 ℃, then the centrifugation is carried out for 10min under the condition of 6000r/min, and the precipitate is discarded after the centrifugation, so as to obtain the protein extracting solution.
According to the preferred embodiment of the present invention, the preparation method of the magnetic biochar is as follows:
step 1: preparation of mushroom dreg biochar
Taking mushroom dregs as a raw material, drying and sieving the mushroom dregs, mixing and activating the mushroom dregs with potassium carbonate, and pyrolyzing the mixture under the inert atmosphere condition to obtain mushroom dreg biochar;
step 2: preparation of magnetic biochar
To contain Fe3+And Fe2+Adding the fungus dreg biochar prepared in S1 into the ferric salt solution, stirring uniformly, adding concentrated ammonia water into the ferric salt solution at a constant speed, stirring and reacting at a constant temperature of 50-65 ℃, and carrying out the reaction process under the protection of inert atmosphere; after the reaction is finished, separating solid from the reaction liquid by adopting a permanent magnet, washing the solid by pure water and ethanol, drying, grinding and sieving to obtain powdery nano magnetic biochar;
and step 3: modification of magnetic biochar
Modifying the magnetic biochar prepared in the step 2 by adopting a silane coupling agent containing amino to obtain amino modified magnetic biochar nanoparticles;
and 4, step 4: preparation of magnetic biochar immobilized enzyme
And (3) adding the amino modified magnetic biochar nanoparticles obtained in the step (3) into a glutaraldehyde solution, carrying out oscillation reaction at normal temperature, washing a reaction product with a phosphate buffer solution, removing a supernatant after washing, adding a solution of a complex enzyme, carrying out oscillation fixation at 25-35 ℃ for a preset time, separating solids by using a permanent magnet, and carrying out deionized water washing to obtain the magnetic biochar immobilized enzyme.
According to the preferred embodiment of the invention, step 1, the mushroom dregs are penicillin mushroom dregs; the method for preparing the mushroom dreg biochar comprises the following steps: drying the penicillin fungi residues, sieving the penicillin fungi residues with a sieve of 80-150 meshes, taking the fungi residues and a potassium carbonate activating agent, mixing the fungi residues and the potassium carbonate activating agent in a proportion of 1: 1-1.2, placing the mixture into a high-purity nitrogen atmosphere, heating the mixture to 550 ℃ and 650 ℃ at a speed of 8-10 ℃/min, and pyrolyzing the mixture for 1-2 hours to obtain the bacterial residue biochar.
Preferably, the mushroom dregs include, but are not limited to, streptomycin mushroom dregs, terramycin mushroom dregs, penicillin mushroom dregs. Preferably, the heating rate is 10 ℃/min, and the pyrolysis temperature is 600 ℃.
According to a preferred embodiment of the present invention, step 2, Fe is in the iron salt solution3+And Fe2+The molar ratio of (A) to (B) is 2: 0.9-1.05.
Preferably, the iron salt solution is FeCl3And FeSO4The mixed solution of (1); the method for preparing the magnetic biochar comprises the following steps: introducing nitrogen into the reactor to exhaust air, adding ultrapure water for removing oxygen, and adding FeCl3Or hydrates thereof with FeSO4Or adding the hydrate into a reactor, stirring until the hydrate is completely dissolved and mixed, adding the bacteria residue organisms, continuously stirring, then adding concentrated ammonia water into the mixed solution by using a constant flow pump, stirring and reacting for 1.5-2h under the constant temperature condition of a water bath at 58-62 ℃, cooling after the reaction is finished, separating the product by using a permanent magnet, washing the product by using ultrapure water for 4-5 times, washing by using absolute ethyl alcohol for 2-4 times, drying in a vacuum drying box at 55-65 ℃ to obtain blocky magnetic biochar, crushing the blocky magnetic biochar in an oxygen-free environment, and sieving by using a sieve of 80-120 meshes to obtain the powdery nano magnetic biochar material.
According to a preferred embodiment of the present invention, in step 3, the amino-containing silane coupling agent is 3-Aminopropyltriethoxysilane (APTES), and the modification method is:
adding magnetic biochar nanoparticles into a reactor filled with ethanol, carrying out ultrasonic pretreatment for 10-30min, placing the reactor in a constant-temperature environment, slowly adding unequal amounts of 3-aminopropyltriethoxysilane in portions under the protection of nitrogen, continuously stirring, and heating to be less than or equal to 70 ℃ for refluxing for a period of time to obtain amino-modified magnetic biochar nanoparticles; then, absolute ethyl alcohol is used for ultrasonic cleaning for 3 times, the amino modified magnetic biochar nano particles are separated from the reactor by adopting a permanent magnet, and the obtained product is dried for later use.
According to a preferred embodiment of the present invention, in step 4, the preparation method of the magnetic biochar immobilized enzyme comprises: taking amino modified magnetic biochar nanoparticles, adding a glutaraldehyde solution with the concentration of 2.5-3.5%, wherein the proportion of the glutaraldehyde solution to the amino modified magnetic biochar nanoparticles is that 8-12mL of glutaraldehyde solution is correspondingly added to each 1g of amino modified magnetic biochar nanoparticles; oscillating and reacting for 1-2h at normal temperature, washing a reaction product by using a phosphate buffer solution, removing a supernatant after washing, adding a complex enzyme solution, oscillating and fixing for 4-6h at 30 ℃, separating solids by using a permanent magnet, and washing by using deionized water until no free enzyme exists, thereby preparing the magnetic biochar immobilized enzyme.
(III) advantageous effects
The invention obtains the optimal complex enzyme (the lysozyme and the neutral protease are compounded) through screening, the optimal complex enzyme is used as the enzyme for extracting the protein of the antibiotic residues, and the enzyme is immobilized by taking the magnetic biochar as a carrier, so that the antibiotic residues are recycled and reduced, and the antibiotic residue in the residues is reduced. Tests show that the effect of the lysozyme and the neutral protease compounded as antibiotic bacteria residue treatment enzymes is better than that of a single enzyme, which shows that a certain synergistic effect exists between the two enzymes, and the protein in the bacteria residue can be effectively extracted.
When the magnetic biochar is prepared, the antibiotic mushroom residues are used as raw materials and pyrolyzed with a potassium carbonate activating agent in an inert atmosphere to prepare the porous biochar. The antibiotic fungi residues have the characteristics of small particles and large specific surface area, and the magnetic biochar prepared from the fungi residues has the nanoscale pore diameter of 50-200nm and the surface area of 1761.11m2The bacterial slag has strong loading capacity, meanwhile, the bacterial slag is greatly reduced in the high-temperature pyrolysis process, antibiotics in the bacterial slag are carbonized and decomposed by pyrolysis, thalli are killed, and the harmless treatment of the bacterial slag is realized.
According to the magnetic biochar immobilized enzyme disclosed by the invention, the carrier material has no negative influence on the enzyme activity, the enzyme performance is stable, and streptomycin fungi residues are treated by the magnetic biochar immobilized enzyme; after 10 times of recycling, the relative activity can still reach 80 percent. The magnetic biochar immobilized enzyme composite material prepared by the invention has the advantages of high stability, easiness in recovery and separation, larger specific surface area, good pore volume adsorption, recyclability and the like. As a carrier for fixing the enzyme, the magnetic biochar has good compatibility with the enzyme, does not influence the activity of the enzyme, can be repeatedly utilized, and has enzyme loading capacity of 40 percent.
The magnetic biochar immobilized enzyme can be repeatedly used, can adapt to the treatment environment compared with free enzyme, has more stable performance, can be separated and recovered by utilizing an external magnetic field after being used, is more favorable for effectively separating the immobilized enzyme from the mushroom dregs, continuously treats the mushroom dregs, improves the utilization efficiency of the magnetic biochar immobilized enzyme, and greatly reduces the capital cost.
Drawings
FIG. 1 shows the effect of different enzyme ratios of neutral protease and lysozyme on the extraction of protein from mushroom dregs.
FIG. 2 is a first scanning electron micrograph of the magnetic biochar prepared in example 3 of the present invention.
FIG. 3 is a scanning electron micrograph II of the magnetic biochar prepared in example 3 of the present invention.
FIG. 4 is a Fourier transform infrared spectrum of magnetic biochar prepared in example 3 of the invention.
FIG. 5 is a scanning electron microscope image of the immobilized complex enzyme on the magnetic biochar carrier prepared in example 3 of the present invention.
Detailed Description
For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.
Example 1
The five enzymes are compounded according to different mass ratios, and the extraction rates of different complex enzymes on the streptomycin residue protein are respectively tested. The five enzymes are neutral protease, alkaline protease, amylase, lipase and lysozyme. The test method comprises the following steps:
adjusting the pH value of 100ml of streptomycin residue liquid with the water content of 95.5 percent to 8, adding 0.2g of complex enzyme, keeping the reaction temperature below 50 ℃, using a constant-temperature shaking table for 120r/min, continuously stirring and hydrolyzing for 7h, separating and recovering the immobilized enzyme in an external magnetic field after the reaction is finished, inactivating the enzyme for 15min under the condition of 100 ℃ water bath, then centrifuging for 10min under 5000r/min, and discarding the precipitate after the centrifugation to obtain a protein extracting solution; and (3) diluting the protein extracting solution, adding a forskolin phenol reagent, measuring absorbance at the wavelength of 650nm after the reaction is finished, calculating the content of the protein in the extracting solution according to a drawn standard curve, and calculating the extraction rate of the immobilized complex enzyme on the protein in the mushroom dregs through the content of crude protein in the antibiotic mushroom dregs.
| Proportioning | 1:2 | 1:1 | 2:1 |
| Neutral protease + alkaline protease | 31％ | 40％ | 35％ |
| Neutral protease and amylase | 37％ | 28％ | 34％ |
| Neutral protease + |
36％ | 30％ | 33％ |
| Neutral protease + lysozyme | 64％ | 56％ | 50％ |
| Alkaline protease + |
36％ | 41％ | 33％ |
| Alkaline protease + lipase | 48％ | 45％ | 40％ |
| Alkaline protease + lysozyme | 42％ | 46％ | 44％ |
| Amylase + Lipase | 37％ | 32％ | 33％ |
| Amylase + lysozyme | 32％ | 30％ | 29％ |
| Lipase + lysozyme | 31％ | 29％ | 32％ |
As can be seen from the table above, the lysozyme and the neutral protease are compounded, so that the extraction effect on the streptomycin fungi residue protein is optimal. Meanwhile, the compounding ratio of lysozyme and neutral protease is adjusted, and the protein content in the streptomycin residue enzymolysis supernate is respectively measured. The results are shown in FIG. 1. Therefore, the complex enzyme is obtained by lysozyme and neutral protease according to the mass ratio of 2:1, and the extraction efficiency of the protein in the antibiotic fungi residues is optimal.
The enzyme in the following magnetic biochar immobilized enzyme is lysozyme and neutral protease which are compounded according to the mass ratio of 2: 1.
Example 2
Compounding lysozyme and neutral protease according to the mass ratio of 2:1, and optimizing the extraction conditions of the compound enzyme on streptomycin fungi residue protein. And (3) taking the streptomycin residues with a certain water content, adjusting conditions such as pH, enzymolysis temperature and enzymolysis time, and testing the extraction rate of protein in the streptomycin residues under different conditions.
Setting the reaction temperature at 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C; the reaction pH is 5, 6, 7, 8 and 9; the enzymolysis time is selected from 1h, 2h, 3h, 4h, 5h and 6h to investigate the influence of various factors on the extraction rate of the protein in the mushroom dregs.
Finally, determining the complex enzyme, wherein the optimal reaction conditions are that the pH is 7, the reaction temperature is 50 ℃, the reaction time is 2h, and the rotating speed of a shaking table is more suitable at 120 r/min.
Therefore, the antibiotic bacterial residue hydrolysis conditions are as follows: adjusting the pH value of the antibiotic fungi residues to 7, adding a proper amount of magnetic biochar immobilized enzyme, reacting in a constant-temperature shaking table at 120r/min, keeping the reaction temperature at 50 ℃, and performing enzymolysis for 2 hours.
Example 3
The embodiment provides a preparation method of a magnetic biochar immobilized enzyme composite material, which comprises the following steps:
(1) and (3) mixing the dried and sieved penicillin fungi residue with potassium carbonate according to the mass ratio of 1:1, mixing, activating, drying to constant weight, placing in a tubular furnace, heating to 600 ℃ at the speed of 10 ℃/min under the protection of nitrogen, pyrolyzing for 2h, then carrying out acid washing and water washing to obtain a product, and drying in an oven to obtain the mushroom residue biochar.
(2) 300ml of ultrapure water was taken in a 500ml three-necked flask, and nitrogen gas was introduced thereto at room temperature for 30min while stirring to exhaust the air in the flask. 4.4903g of FeCl were weighed3.6H2O and 2.3074gFeSO4.7H2And O, uniformly mixing in the three-neck flask, adding 6g of mushroom dreg biochar, magnetically stirring for 30 minutes, dripping 30ml of concentrated ammonia water into the mixed solution through a constant flow pump, generating a large amount of black precipitates immediately, continuously stirring in a water bath kettle at the temperature of 60 ℃ for reaction for 2 hours, and carrying out the whole experimental process under the protection of nitrogen. Inverse directionAfter completion of the reaction, the mixture was stirred and cooled, and separated with a magnet. Cleaning with ultrapure water and anhydrous ethanol, drying in a vacuum drying oven at 60 deg.C for 20 hr to obtain dried block-shaped magnetic biological carbon, pulverizing in a sealed bag with air removed, and sieving with 100 mesh sieve to obtain powdered magnetic biological carbon material.
As shown in fig. 2-3, are scanning electron micrographs of the prepared magnetic biochar. As can be seen from the figure, the surface of the prepared biomass charcoal has a large number of pores. The surface is distributed with rich and rough pore structure. The aperture is about 50 nm-200 nm, the aperture distribution is relatively uniform, the size is relatively uniform, and the surface area is large. The multipoint specific surface area of the carrier prepared in the figure 2 is 1761.11m2A pore area of 1577.33m2A micropore volume of 0.62 m/g3(ii)/g; the multipoint specific surface area of the carrier prepared in the figure 3 is 1217.04m2A pore area of 1148.46m2A micropore volume of 0.46 m/g3/g
As shown in fig. 4, is an infrared spectrum of the prepared magnetic biochar. As can be seen from the figure, the content of N element in the magnetic biochar prepared by the scheme reaches 6.95 percent, wherein the content of N is higher than that of common commercial activated carbon.
(3) And (3) putting 0.5g of magnetic biochar nanoparticles into a three-neck flask, adding 40mL of ethanol, and performing ultrasonic pretreatment for 30 min. And (2) placing the three-neck flask in a constant-temperature water bath kettle, slowly adding unequal amounts of 3-Aminopropyltriethoxysilane (APTES) for multiple times under the protection of nitrogen, continuously mechanically stirring, and reacting at a certain temperature for a period of time to obtain the amino modified magnetic biochar nanoparticles. Then, the carrier was separated from the flask with a permanent magnet after 3 ultrasonic washes with absolute ethanol for 10min each time to ensure that 3-aminopropyltriethoxysilane was washed clean. The obtained amino modified magnetic biochar nano-particles (MBC-NH)2) And vacuum drying at 80 deg.C.
(4) Mixing lysozyme and neutral protease in a ratio of 2:1, taking 0.2g of nano magnetic carrier, adding 2mL of 3% glutaraldehyde solution, and oscillating and reacting for 1h at normal temperature. Washing with phosphate buffer solution for 3 times, removing supernatant, adding 20mL of complex enzyme solution, oscillating and fixing for 4h at 30 ℃, separating the mixture with a permanent magnet, and repeatedly washing with deionized water until no enzyme is washed out to obtain the magnetic biochar immobilized complex enzyme, wherein FIG. 5 is a scanning electron microscope image of the magnetic biochar immobilized complex enzyme, the image shows that the carrier has a plurality of pore structures, a plurality of spherical substances on micropores are immobilized enzymes, and a part of the carrier is agglomerated, which is probably a cross-linking reaction on the surface of the carrier after amino modification in the immobilization process.
Example 4
In this example, the penicillin mushroom dregs in the step (1) is changed into streptomycin mushroom dregs on the basis of the example 3.
Example 5
In this example, on the basis of example 3, the pyrolysis temperature in the step (1) was adjusted to 650 ℃ and the temperature increase rate was 8 ℃/min.
Example 6
In this example, on the basis of example 3, 30ml of concentrated ammonia water in step (2) was added to the mixed solution quickly at a time.
Example 7
In this example, 3-Aminopropyltriethoxysilane (APTES) in the step (3) was changed to KH-570 based on example 3.
Example 7
In this example, on the basis of example 2, lysozyme and alkaline protease in step (4) are mixed in a ratio of 2:1 is mixed as complex enzyme, and the dosage is equal to the total amount of the complex enzyme in the example 2.
Example 9
In this example, on the basis of example 3, the lipase and the neutral protease in step (4) are mixed according to the following ratio of 2:1 is mixed as complex enzyme, and the dosage is equal to the total amount of the complex enzyme in the example 2.
Example 10
In this example, on the basis of example 3, the lysozyme in step (4) is removed, only the neutral protease is retained, and the amount of the neutral protease is equal to the total amount of the complex enzyme in example 2.
The treatment method of the streptomycin bacterial residues by adopting the magnetic biochar immobilized enzyme composite material prepared in the embodiment 2-9 comprises the following steps:
adjusting the pH value of 100ml of mushroom residue liquid with the water content of 95.5 percent to 7, adding 0.25g of magnetic biochar immobilized complex enzyme, keeping the reaction temperature at 50 ℃, using a constant-temperature shaking table for 120r/min, continuously stirring and hydrolyzing for 2h, separating and recovering the immobilized enzyme in an external magnetic field after the reaction is finished, inactivating the enzyme for 15min under the condition of 100 ℃ water bath, centrifuging for 10min at 5000r/min, and discarding the precipitate after the centrifugation to obtain a protein extracting solution; and (3) diluting the protein extracting solution, adding a forskolin phenol reagent, measuring the absorbance at the wavelength of 650nm after the reaction is finished, calculating the protein content in the extracting solution according to a drawn standard curve, and obtaining the extraction rate of the immobilized complex enzyme on the protein in the mushroom dregs through the content of crude protein in the antibiotic mushroom dregs. At the same time, the treatment with unfixed free enzyme was carried out in the same manner as described above (lysozyme mixed with neutral protease at 2:1 by mass) as a reference result.
The activity of the first extraction of the protein from the mushroom dregs using the immobilized enzyme was regarded as 100%, and the activity of each of the following activities was compared with each other, and similarly expressed as the residual activity. After each reaction is finished, magnetically separating the reaction system, and cleaning the immobilized enzyme catalyst for further use in the next protein extraction reaction system.
The relationship between the enzyme activity (%) and the number of cycles of the enzyme-treated streptomycin dregs prepared in examples 3 to 10 is shown in the following table.
| Number of |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Example 3 | 100 | 98.5 | 95.2 | 94.6 | 90.8 | 88.6 | 87.5 | 84.7 | 82.9 | 80.5 |
| Example 4 | 99.5 | 98.2 | 94.5 | 93.2 | 88.7 | 85.6 | 84.3 | 83.8 | 81.8 | 79.4 |
| Example 5 | 95.6 | 94.1 | 90.9 | 89.4 | 87.1 | 84.0 | 82.4 | 81.7 | 80.2 | 78.6 |
| Example 6 | 94.2 | 92.2 | 90.5 | 88.4 | 86.2 | 84.8 | 83.1 | 79.9 | 78.1 | 78.9 |
| Example 7 | 99.8 | 98.4 | 95.1 | 94.8 | 90.2 | 88.7 | 87.2 | 84.4 | 83.2 | 80.6 |
| Practice ofExample 8 | 90.8 | 89.0 | 86.9 | 82.6 | 80.4 | 78.1 | 76.5 | 74.6 | 73.4 | 72.1 |
| Example 9 | 90.0 | 88.4 | 83.5 | 80.0 | 79.5 | 77.2 | 75.6 | 72.1 | 71.0 | 70.0 |
| Example 10 | 91.2 | 89.1 | 87.2 | 85.6 | 83.4 | 80.9 | 78.4 | 76.2 | 74.8 | 73.2 |
| Free enzyme | 99.5 | —— | —— | —— | —— | —— | —— | —— | —— | —— |
By analyzing the indexes, the method fully proves that the magnetic biochar immobilized complex enzyme retains the activity of the enzyme and improves the stability of the enzyme. The magnetic charcoal as a carrier is more beneficial to the immobilization of the enzyme and the effective separation of the enzyme from the mushroom dregs, improves the utilization efficiency of the immobilized enzyme, greatly reduces the capital cost, and particularly has the highest enzymolysis efficiency and better stability of the immobilized enzyme prepared in the embodiment 3 and the embodiment 7.
The magnetic biochar is used as a carrier of immobilized enzyme, has the characteristics of large specific surface area, porosity and surface modification, and overcomes the defect of magnetic Fe3O4The nano particles are easy to agglomerate, the specific surface area is small, and the like, and the bacteria residues are used as raw materials to prepare the magnetic biochar, the raw materials are sufficient and easy to obtain, and the magnetic biochar has the same magnetic characteristic, can be separated and recovered by a method of an external magnetic field, and improves the utilization efficiency of enzyme.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for separating protein from antibiotic fungi residues is characterized in that the method uses magnetic biochar immobilized enzyme to hydrolyze the antibiotic fungi residues and separate the protein from the antibiotic fungi residues; the magnetic biochar immobilized enzyme is a complex enzyme compounded by lysozyme and neutral protease, and the magnetic biochar is used as a carrier on which the lysozyme and the neutral protease are fixed.
2. The method according to claim 1, wherein the complex enzyme is prepared by compounding lysozyme and neutral protease according to a mass ratio of 1: 1-2: 1.
3. The method as claimed in claim 2, wherein the complex enzyme is prepared by mixing lysozyme and neutral protease according to the mass ratio of 2: 1.
4. The method according to claim 2, wherein the mass ratio of the complex enzyme to the magnetic biochar is 1: 2-7.
5. The method as claimed in claim 1, wherein the pH of the antibiotic residue liquid is adjusted to 7-9, a magnetic biochar immobilized complex enzyme is added, the reaction temperature is kept at 50-55 ℃, a constant temperature shaking table is used for 60-150r/min, continuous stirring and enzymolysis are carried out for 2-3h, the immobilized enzyme is separated and recovered in an external magnetic field after the reaction is finished, the enzyme is inactivated for 10-20min under the condition of water bath at 100 ℃, then the solution is centrifuged for 10min at 4000-6000r/min, and the precipitate is discarded after the centrifugation, so as to obtain the protein extracting solution.
6. The method according to claim 2 or 3, wherein the magnetic biochar is prepared by the following method:
step 1: preparation of mushroom dreg biochar
Taking mushroom dregs as a raw material, drying and sieving the mushroom dregs, mixing and activating the mushroom dregs with potassium carbonate, and pyrolyzing the mixture under the inert atmosphere condition to obtain mushroom dreg biochar;
step 2: preparation of magnetic biochar
To contain Fe3+And Fe2+Adding the fungus dreg biochar prepared in S1 into the ferric salt solution, stirring uniformly, adding concentrated ammonia water into the ferric salt solution at a constant speed, stirring and reacting at a constant temperature of 50-65 ℃, and carrying out the reaction process under the protection of inert atmosphere; after the reaction is finished, separating solid from the reaction liquid by adopting a permanent magnet, washing the solid by pure water and ethanol, drying, grinding and sieving to obtain powdery nano magnetic biochar;
and step 3: modification of magnetic biochar
Modifying the magnetic biochar prepared in the step 2 by adopting a silane coupling agent containing amino to obtain amino modified magnetic biochar nanoparticles;
and 4, step 4: preparation of magnetic biochar immobilized enzyme
And (3) adding the amino modified magnetic biochar nanoparticles obtained in the step (3) into a glutaraldehyde solution, carrying out oscillation reaction at normal temperature, washing a reaction product with a phosphate buffer solution, removing a supernatant after washing, adding a solution of a complex enzyme, carrying out oscillation fixation at 25-35 ℃ for a preset time, separating solids by using a permanent magnet, and carrying out deionized water washing to obtain the magnetic biochar immobilized enzyme.
7. The method of claim 6, wherein in step 1, the mushroom dregs are penicillin mushroom dregs; the method for preparing the mushroom dreg biochar comprises the following steps: drying the penicillin fungi residues, sieving the penicillin fungi residues with a sieve of 80-150 meshes, taking the fungi residues and a potassium carbonate activating agent, mixing the fungi residues and the potassium carbonate activating agent in a proportion of 1: 1-1.2, placing the mixture into a high-purity nitrogen atmosphere, heating the mixture to 550 ℃ and 650 ℃ at a speed of 8-10 ℃/min, and pyrolyzing the mixture for 1-2 hours to obtain the bacterial residue biochar.
8. The method of claim 6, wherein the method of preparing the magnetic biochar is: introducing nitrogen into the reactor to exhaust air, adding ultrapure water for removing oxygen, and adding FeCl3Or hydrates thereof with FeSO4Or its hydrate is added into a reactor, Fe is dissolved in iron salt solution3+And Fe2+The molar ratio of (A) to (B) is 2: 0.9-1.05; stirring to dissolve completely, adding bacteria residue, and stirringAdding concentrated ammonia water into the mixed solution by using a constant flow pump, stirring and reacting for 1.5-2h under the constant temperature condition of a water bath at 58-62 ℃, cooling after the reaction is finished, separating a product by using a permanent magnet, washing the product for 4-5 times by using ultrapure water, washing the product for 2-4 times by using absolute ethyl alcohol, drying the product in a vacuum drying oven at 55-65 ℃ to obtain blocky magnetic biochar, and crushing the blocky magnetic biochar in an oxygen-free environment and sieving the blocky magnetic biochar by using a sieve of 80-120 meshes to obtain a powdery nano magnetic biochar material.
9. The method according to claim 6, wherein in step 3, the amino group-containing silane coupling agent is 3-aminopropyltriethoxysilane, and the modification method is:
adding magnetic biochar nanoparticles into a reactor filled with ethanol, carrying out ultrasonic pretreatment for 10-30min, placing the reactor in a constant-temperature environment, slowly adding unequal amounts of 3-aminopropyltriethoxysilane in portions under the protection of nitrogen, continuously stirring, and heating to be less than or equal to 70 ℃ for refluxing for a period of time to obtain amino-modified magnetic biochar nanoparticles; then, absolute ethyl alcohol is used for ultrasonic cleaning for 3 times, the amino modified magnetic biochar nano particles are separated from the reactor by adopting a permanent magnet, and the obtained product is dried for later use.
10. The method of claim 6, wherein in the step 4, the magnetic biochar immobilized enzyme is prepared by the following steps: taking amino modified magnetic biochar nanoparticles, adding a glutaraldehyde solution with the concentration of 2.5-4.5%, wherein the proportion of the glutaraldehyde solution to the amino modified magnetic biochar nanoparticles is that 8-12mL of glutaraldehyde solution is correspondingly added to each 1g of amino modified magnetic biochar nanoparticles; oscillating and reacting for 1-2h at normal temperature, washing a reaction product by using a phosphate buffer solution, removing a supernatant after washing, adding a complex enzyme solution, oscillating and fixing for 4-6h at 30 ℃, separating solids by using a permanent magnet, and washing by using deionized water until no free enzyme exists, thereby preparing the magnetic biochar immobilized enzyme.
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