A decolorizing agent for polylysine and its preparation method and application
By immobilizing laccase on β molecular sieve, the decolorizer for polylysine is prepared, which solves the problem of difficult removal of pigment substances during the polylysine separation process, and efficient pigment removal and reusable decolorizer are achieved, reducing costs.
Patent Information
- Application Number
- CN202411888514.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-20
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art is difficult to effectively remove pigment substances produced during polylysine separation, affecting the purity and quality of the product.
A decolorizing agent for polylysine is prepared by immobilizing laccase on a beta molecular sieve. The method of preparing the decolorizer includes adding laccase and glycerol to a phosphate buffer, mixing evenly, then shaking and mixing with a β-molecular sieve, solidifying and drying, and obtaining an immobilized laccase decolorizer.
This decolorizing agent significantly improves the decolorization effect of polylysine, the pigment removal rate can reach more than 95%, and the decolorization reaction can be carried out at room temperature, and can be reused, reducing costs.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biomedicine, in particular to a decolorizing agent for polylysine and a preparation method and application thereof. Background Art
[0002] Polylysine is a biological food preservative with broad-spectrum antibacterial effects, which has the advantages of good water solubility, wide pH range, strong thermal stability, low usage, broad antibacterial spectrum and high safety. Currently, polylysine has been used in countries and regions such as Japan, South Korea, the United States and Europe.
[0003] At present, the separation and purification of polylysine is mainly achieved in China through ion exchange adsorption and multi-stage membrane separation. However, the pigment substances derived from organic nitrogen sources, Maillard reaction products during sterilization, and the pigment substances secreted by the producing bacteria themselves cannot be effectively removed in the above extraction process, thereby affecting the sensory properties of polylysine products and limiting the improvement of purity and quality. Therefore, it is of great significance to provide an effective decolorizing agent and decolorizing method for polylysine. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a decolorizing agent for polylysine with good decolorizing effect; the present invention also provides a preparation method of the decolorizing agent for polylysine; the present invention also provides an application of the decolorizing agent for polylysine.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: immobilizing laccase on beta molecular sieve.
[0006] Furthermore, the mass ratio of the laccase to the beta molecular sieve is 40-60 mg / g.
[0007] In order to solve the above technical problems, the technical scheme adopted by the method of the present invention includes the following steps: adding laccase and glycerol to a phosphate buffer and mixing them evenly to form a mixed solution, wherein the laccase concentration is 1-1.5 mg / ml, the glycerol addition amount is 15%-20 vol%, and the pH of the phosphate buffer is 2-4; placing β molecular sieve into the mixed solution and oscillating for 12-24 hours, wherein the mass ratio of laccase to β molecular sieve is 40-60 mg / g; then separating to obtain a solid, and after drying, the decolorizing agent can be obtained.
[0008] Furthermore, the silicon-aluminum ratio of the β molecular sieve is 35-60.
[0009] Furthermore, the concentration of the phosphate buffer is 0.2 mol / L.
[0010] In order to solve the above technical problems, the technical solution adopted by the present invention is: adjusting the pH of the analytical solution containing polylysine to 4.0-6.0, adding the decolorizing agent, stirring sufficiently and filtering to obtain a decolorizing solution.
[0011] Furthermore, the amount of the decolorizing agent added is 10 to 20 g / L.
[0012] Furthermore, the stirring temperature is 20-30° C., and the stirring time is 6-8 hours.
[0013] The beneficial effects of adopting the above technical solution are: The laccase described in the present invention is a polyphenol oxidase that can catalyze phenols and aromatic amines and is widely used in the fields of pulp bleaching, sewage treatment, beverage decolorization, etc. The beta molecular sieve has good thermal stability and a large specific surface area, and has good biocompatibility with biological enzymes. Glycerol is a polyhydroxy compound, which can increase the stability of laccase as an additive. At the same time, during the immobilization process, glycerol can increase the compatibility between the immobilized carrier and the laccase, so that the laccase is more evenly distributed on the carrier surface, improve the relative enzyme activity and activity recovery rate of the immobilized laccase, and also help maintain the conformational integrity of the immobilized laccase, and prevent the enzyme molecules from being deformed or inactivated during the immobilization process.
[0014] The present invention uses glycerol as an additive and beta molecular sieve as a carrier material to immobilize laccase, and has a significant decolorization effect, with a pigment removal rate of more than 95%. At the same time, the decolorizer can perform a decolorization reaction at room temperature and can be reused, greatly reducing costs. DETAILED DESCRIPTION
[0015] The present invention is further described in detail below in conjunction with specific implementation modes.
[0016] The decolorizing agent for polylysine is laccase immobilized on beta molecular sieve, wherein the mass ratio of laccase to beta molecular sieve is 40-60 mg / g.
[0017] The preparation method of a decolorizing agent for polylysine comprises the following steps: (1) adding laccase and glycerol into a phosphate buffer and mixing them uniformly to form a mixed solution, wherein the laccase addition concentration is 1 to 1.5 mg / ml, the glycerol addition amount is 15% to 20% of the volume of the phosphate buffer, and the pH of the phosphate buffer is 2 to 4; (2) adding β molecular sieve into the mixed solution and oscillating the mixture at an oscillator speed of 160 to 180 r / min for 12 to 24 hours, wherein the mass ratio of laccase to β molecular sieve is 40 to 60 mg / g, and the silicon-aluminum ratio of the β molecular sieve is 35 to 60; (3) After shaking, the mixed liquid is separated to obtain a solid, which is then dried at low temperature to obtain the decolorizing agent, i.e., β-molecular sieve-immobilized laccase.
[0018] The application of the decolorizing agent for polylysine includes the following steps: adjusting the pH of the analytical solution containing polylysine to 4.0-6.0 with hydrochloric acid, and the concentration of the analytical solution is 30g / L-50g / L; adding the decolorizing agent, the amount of the decolorizing agent is 10-20g / L, stirring at a stirring temperature of 20-30°C and then filtering, the stirring speed is 40-60r / min, and the stirring time is 6-8h, and the decolorizing solution is obtained. After the beta molecular sieve immobilized laccase decolorizing agent is used, it can be washed with 0.2mol / L phosphate buffer and reused. When the decolorizing agent is used for the decolorization of polylysine, the decolorization rate can reach more than 95%, and the decolorization rate can reach more than 90% after 5 times of use.
[0019] In the following examples, the determination method and calculation formula of relative enzyme activity, activity recovery rate and pigment removal rate are as follows: Determination of free enzyme activity: 2,2-azino-bis(3-ethylthiophene-6-sulfonic acid) (ABTS) is used as the substrate, and the quantitatively diluted enzyme solution and 1.0mmol / L ABTS are reacted in equal volumes. The medium used is 0.2mol / L PBS with an appropriate pH value. Determine the absorbance change ΔA at 420nm in the reaction system within 4 minutes. O .
[0020] Determination method of immobilized laccase: take 0.05g immobilized laccase, add 2.5mL buffer solution of corresponding pH value and 2.5mL, 1.0mmol / L ABTS solution, stop the reaction in an ice bath after 15min, centrifuge and filter, and measure the absorbance ΔA1 of the supernatant at 420nm.
[0021] The relative enzyme activity calculation formula is as follows: Relative enzyme activity (%) = (ΔA1 / ΔA O )×100%.
[0022] Determination of activity recovery rate: accurately weigh 1g of free laccase and immobilize it according to the established process. After thorough washing, place it in 50ml buffer and determine the activity E1 of immobilized laccase under the same conditions as above, given the total enzyme activity E0 of free laccase.
[0023] The calculation formula of vitality recovery rate is as follows: Vitality recovery rate (%) = (E 1 / E0)×100%.
[0024] Method for determining pigment removal rate: dilute the sample solution 20 times, determine its absorbance at 410nm, and the decolorization rate is calculated as follows: Decolorization rate (%) = (Ao-Ae) / Ao×100%, where Ao is the absorbance of the sample solution at 410nm before decolorization, and Ae is the absorbance of the sample solution at 410nm after decolorization.
[0025] Determination method of polylysine: Itzhaki method (Itzhaki, 1972) was used. Example 1
[0026] (1) Weigh 10 g of laccase and dissolve it in 10 L of phosphate buffer with a pH of 3 and a concentration of 0.2 mol / L. Add 1.5 L of glycerol to the laccase solution and stir well to obtain a mixed solution.
[0027] (2) Suspend 200 g of β molecular sieve with a silicon-aluminum ratio of 40 in the mixed solution and react in an oscillator at a speed of 160 r / min for 20 h.
[0028] (3) The solid was obtained by centrifugal separation and low-temperature drying to obtain 120 g of β-molecular sieve immobilized laccase decolorizing agent with a relative enzyme activity of 68% and an activity recovery rate of 44%.
[0029] (4) 5 L of analytical solution containing polylysine was measured, the concentration of the analytical solution was 40 g / L, the pH was adjusted to 5.0 with hydrochloric acid, 75 g of the above-mentioned β-molecular sieve immobilized laccase decolorizing agent was added, and after being stirred for 6 hours at a temperature of 25°C and a stirring speed of 55 rpm, a decolorizing solution was obtained, and the pigment removal rate was 95.4%. Example 2
[0030] (1) Weigh 24 g of laccase and dissolve it in 16 L of phosphate buffer with a pH of 3.5 and a concentration of 0.2 mol / L; add 3 L of glycerol to the laccase solution and stir well to obtain a mixed solution.
[0031] (2) Suspend 400 g of β molecular sieve with a silicon-aluminum ratio of 40 in the mixed solution and react on an oscillator at a speed of 180 r / min for 24 h.
[0032] (3) The solid was obtained by centrifugal separation and low-temperature drying to obtain 280 g of β-molecular sieve immobilized laccase decolorizing agent with a relative enzyme activity of 72% and an activity recovery rate of 50%.
[0033] (4) 10 L of analytical solution containing polylysine was measured, the concentration of the analytical solution was 50 g / L, the pH was adjusted to 5.8 with hydrochloric acid, 200 g of the above-mentioned β-molecular sieve immobilized laccase decolorizing agent was added, and after being fully stirred at a temperature of 30°C and a stirring speed of 60 rpm for 8 h, a decolorizing solution was obtained, and the pigment removal rate was 98.7%. Example 3
[0034] (1) Weigh 30 g of laccase and dissolve it in 20 L of phosphate buffer with a pH of 2 and a concentration of 0.2 mol / L; add 3.6 L of glycerol to the laccase solution and stir well to obtain a mixed solution.
[0035] (2) 750 g of β molecular sieve with a silicon-aluminum ratio of 60 was suspended in the mixed solution and reacted in an oscillator at a speed of 170 r / min for 12 h.
[0036] (3) The solid was obtained by centrifugal separation and low-temperature drying to obtain 580 g of β-molecular sieve immobilized laccase decolorizing agent with a relative enzyme activity of 65% and an activity recovery rate of 47%.
[0037] (4) 10 L of analytical solution containing polylysine was measured, the concentration of the analytical solution was 45 g / L, the pH was adjusted to 6.0 with hydrochloric acid, 180 g of the above-mentioned β-molecular sieve immobilized laccase decolorizing agent was added, and after being stirred for 7 h at a temperature of 20°C and a stirring speed of 50 rpm, a decolorizing solution was obtained, and the pigment removal rate was 97.2%. Example 4
[0038] (1) Weigh 24 g of laccase and dissolve it in 20 L of phosphate buffer with a pH of 4 and a concentration of 0.2 mol / L; add 4 L of glycerol to the laccase solution and stir well to obtain a mixed solution.
[0039] (2) 480 g of β molecular sieve with a silicon-aluminum ratio of 35 was suspended in the mixed solution and reacted on an oscillator at a speed of 170 r / min for 17 h.
[0040] (3) After centrifugal separation, the solid was obtained, and after low-temperature drying, 370 g of β molecular sieve immobilized laccase decolorizer was obtained. The relative enzyme activity was 70%, and the activity recovery rate was 45%.
[0041] (4) 10 L of analytical solution containing polylysine was measured, the concentration of the analytical solution was 30 g / L, the pH was adjusted to 4.0 with hydrochloric acid, 100 g of the above-mentioned β-molecular sieve immobilized laccase decolorizing agent was added, and after being fully stirred at a temperature of 25°C and a stirring speed of 40 rpm for 7 h, a decolorizing solution was obtained, and the pigment removal rate was 96.8%. Example 5
[0042] After the used β molecular sieve immobilized laccase decolorizer in Example 2 was washed with 0.2 mol / L phosphate buffer, the same concentration and volume of feed solution as in Example 2 was added again and decolorized under the same conditions. A total of 5 repeated experiments were performed, and the results are shown in Table 1.
[0043] Table 1: Pigment removal rate after repeated use
[0044] As shown in Table 1, the decolorization rate of polylysine can still reach more than 90% after repeated use of the β molecular sieve immobilized laccase decolorizer for decolorization of polylysine for 5 times, the relative enzyme activity of the decolorizer is 52%, and the activity recovery rate is 39%.
[0045] Comparative Example 1: Weigh 10g of laccase, dissolve it in 10L of 0.2mol / L phosphate buffer with pH=3, and stir evenly. Suspend 200g of β molecular sieve with a silicon-aluminum ratio of 40 on the prepared laccase solution, and react on an oscillator at a speed of 160r / min for 20h. After centrifugal separation, the solid is obtained, and after low-temperature drying, 100g of β molecular sieve immobilized laccase is obtained. The relative enzyme activity of the obtained decolorizer is 45%, and the activity recovery rate is 30%.
[0046] 5 L of analytical solution containing polylysine was measured, the concentration of the analytical solution was 40 g / L, the pH was adjusted to 5.0 with hydrochloric acid, 75 g of β-molecular sieve immobilized laccase obtained in the comparative example was added, and after being fully stirred at a temperature of 25°C and a stirring speed of 55 rpm for 6 hours, a decolorized solution was obtained, and the pigment removal rate was 87.5%.
[0047] It can be seen from Examples 1-5 and the comparative example that the relative enzyme activity and activity recovery rate of the beta molecular sieve immobilized laccase decolorizing agent obtained in the examples are significantly higher than those in the comparative example, and the pigment removal rate when used for decolorization is significantly higher than that in the comparative example.
Claims
1. A decolorizing agent for polylysine, characterized in that: Laccase is immobilized on the β molecular sieve.
2. A decolorizing agent for polylysine according to claim 1, characterized in that: The mass ratio of the laccase to the beta molecular sieve is 40-60 mg / g.
3. The method for preparing a decolorizing agent for polylysine according to claim 1 or 2, characterized in that: The method comprises the following steps: adding laccase and glycerol into a phosphate buffer solution, and mixing them uniformly to form a mixed solution, wherein the laccase concentration is 1-1.5 mg / ml, the glycerol addition amount is 15%-20 vol%, and the pH value of the phosphate buffer solution is 2-4; placing beta molecular sieve into the mixed solution, and oscillating for 12-24 hours, wherein the mass ratio of laccase to beta molecular sieve is 40-60 mg / g; and then separating to obtain a solid, and drying to obtain the decolorizing agent.
4. The method for preparing a decolorizing agent for polylysine according to claim 3, characterized in that: The silicon-aluminum ratio of the beta molecular sieve is 35-60.
5. A method for preparing a decolorizing agent for polylysine according to claim 3 or 4, characterized in that: The concentration of the phosphate buffer is 0.2 mol / L.
6. The use of the decolorizing agent for polylysine according to claim 1, characterized in that: The pH of the analytical solution containing polylysine is adjusted to 4.0-6.0, the decolorizing agent is added, and the solution is filtered after being fully stirred to obtain a decolorizing solution.
7. The use of a decolorizing agent for polylysine according to claim 6, characterized in that: The amount of the decolorizing agent added is 10-20 g / L.
8. The use of a decolorizing agent for polylysine according to claim 6 or 7, characterized in that: The stirring temperature is 20-30° C., and the stirring time is 6-8 hours.