Liquid laccase protective agent and application thereof

Abstract

The invention provides a liquid laccase protective agent and application thereof, and belongs to the technical field of bioengineering. According to the liquid laccase protective agent, a NaOH buffer solution with the pH value of 10.0 is used as a solvent, and solutes comprise trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerin and copper chloride; the liquid laccase protective agent can effectively reduce the enzyme activity loss of laccase in the storage and transportation processes, prolong the storage life of the laccase and improve the application effect of the laccase, so that the application cost of the laccase is reduced, the application of the liquid laccase in the fields of food processing, papermaking industry, textile industry, industrial wastewater treatment and the like is facilitated, and the liquid laccase protective agent has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a liquid laccase protective agent and application thereof. Background Art

[0002] Laccase is an important polyphenol oxidase with a wide range of commercial applications. Laccases can be classified into acidic and alkaline laccases based on their pH adaptability. Compared to acidic laccases, alkaline laccases have higher activity under alkaline conditions and can operate stably over a wider pH range. Therefore, alkaline laccases have better market prospects and huge development potential. Currently, alkaline laccases are widely used in wastewater and soil treatment and remediation, juice clarification, beer classification improvement, and the textile and papermaking industries.

[0003] Since solid laccase has the advantages of simple storage conditions and convenient transportation, the laccase currently in the domestic market is mainly solid laccase. However, solid laccase has the disadvantages of complex production technology, high production cost, and limited application range, which is not conducive to the application of laccase. In contrast, liquid laccase has a simple production process, low production cost, and a wider range of applications, but it is easily affected by environmental factors such as temperature and pH during storage and transportation, which can lead to reduced enzyme activity and thus affect its service life. Therefore, it is necessary to develop a liquid laccase protective agent to reduce the loss of enzyme activity during storage and transportation of laccase and improve its service life. Summary of the Invention

[0004] In response to some deficiencies in the prior art, the present invention provides a liquid laccase protective agent and an application thereof. The liquid laccase protective agent of the present invention uses a NaOH buffer solution with a pH of 10.0 as a solvent, and the solutes include: trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride. The liquid laccase protective agent can effectively reduce the enzyme activity loss of laccase during storage and transportation, improve its storage life and application effect, thereby reducing the application cost of laccase, and is conducive to the application of liquid laccase in food processing, papermaking industry, textile industry and industrial wastewater treatment and other fields, and has good practicality.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention first provides a liquid laccase protective agent, which uses NaOH buffer as a solvent and comprises the following components:

[0007] Trehalose, bovine serum albumin, glycine, surfactant, sodium alginate, glycerol and cupric chloride.

[0008] Preferably, the pH value of the NaOH buffer solution in the liquid laccase protective agent is 10.

[0009] Preferably, the concentration of the NaOH buffer is 1 mM.

[0010] Preferably, the surfactant includes Tween 80, Span 20, Brij 35, NP-40, Triton X-100; preferably Tween 80.

[0011] Preferably, the liquid laccase protective agent comprises the following components: 1-4% (v / v) trehalose, 1-4% (v / v) bovine serum albumin, 1-7% (v / v) glycine, 0.6-1.8% (v / v) Tween 80, 0.5-2.5% (v / v) sodium alginate, 2-14% (v / v) glycerol, and 1-7 mM copper chloride.

[0012] Preferably, the liquid laccase protective agent comprises the following components: 2.5% (v / v) trehalose, 3% (v / v) bovine serum albumin, 3% (v / v) glycine, 1.2% (v / v) Tween 80, 1.5% (v / v) sodium alginate, 10% (v / v) glycerol, and 3 mM copper chloride.

[0013] Preferably, the preparation method of the liquid laccase protective agent comprises: dissolving trehalose, bovine serum albumin, glycine, a surfactant, sodium alginate, glycerol and copper chloride in NaOH buffer one by one to obtain the liquid laccase protective agent.

[0014] The present invention also provides application of the liquid laccase protective agent in improving the storage stability of liquid laccase.

[0015] Preferably, the liquid laccase is an alkaline liquid laccase.

[0016] Preferably, the liquid laccase is derived from animals, plants or microorganisms.

[0017] Preferably, the alkaline liquid laccase comprises laccase CotA.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The invention uses a NaOH buffer solution with a pH of 10.0 as a solvent, and adds trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride thereto to prepare the solution. The trehalose replaces water molecules during the storage of the enzyme to form hydrogen bonds with the enzyme molecules, thereby stabilizing the three-dimensional structure of the enzyme and forming a glassy structure, thereby reducing the contact between the enzyme and the external environment and protecting the enzyme activity. The bovine serum albumin can provide a stable protein environment to protect the enzyme and form a weak interaction with the enzyme to prevent aggregation and precipitation between the enzyme molecules, thereby maintaining the solubility and stability of the enzyme. The glycine acts as a buffer to stabilize the pH value of the solution during the storage of the enzyme and prevent the influence of pH fluctuation on the enzyme activity. The Tween 80 is a surfactant that can wrap the enzyme molecules to form a stable microenvironment, thereby preventing the enzyme from direct contact with air or an interface and reducing interface inactivation. and denaturation; sodium alginate can form a gel network to produce a sieve effect, which can wrap the enzyme molecules, limit their activity, reduce the collision of the enzyme molecules and their direct contact with the external environment, thereby maintaining the activity and stability of the enzyme; glycerol can interact with the water molecules around the enzyme molecules to form hydrogen bonds, thereby maintaining the hydration state of the enzyme and preventing the enzyme structure from being denatured due to dehydration. In addition, glycerol can lower the freezing point of the solution and reduce the damage to the enzyme structure caused by ice crystal formation, especially providing protection during low-temperature freezing; copper chloride stabilizes the activity of laccase by providing copper ions as a cofactor. Copper ions are an important component of the active center of laccase, which can maintain the correct conformation and catalytic ability of the enzyme, help prevent the autocatalytic degradation of the enzyme, and reduce the enzyme inactivation caused by external environmental factors such as pH changes or temperature fluctuations, thereby extending the storage life and stability of laccase.

[0020] The present invention demonstrates, through storage stability experiments, that the liquid laccase protective agent can effectively reduce the loss of liquid laccase enzyme activity, improve its service life and application effect, and reduce the cost of laccase use. Compared to laccase without the addition of the protective agent, the laccase activity increased by 36.1%. The enzyme activity retention rate increased by 19.7% after storage at 4°C for 35 days, improving the storage effect of laccase at 4°C and significantly extending its half-life. The liquid laccase protective agent of the present invention fills a gap in laccase protective agents and has excellent practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The enzymatic activity of alkaline laccase under the protection of different surfactants.

[0022] Figure 2 This is the effect of liquid laccase protective agent on the storage stability of alkaline laccase at 4°C. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the scope of protection of the present invention is not limited thereto. In the following examples, various processes and methods not described in detail are conventional methods well known in the art. The sources, trade names, and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as the sources indicated for the first time unless otherwise specified; the reagents, materials, etc. involved are all obtained for commercial purposes unless otherwise specified.

[0024] The liquid laccase of the present invention is an alkaline liquid laccase; the liquid laccase is derived from animals, plants or microorganisms. The effect of the liquid laccase protective agent is verified below using laccase CotA as an example, wherein the laccase CotA is a laccase recorded on NCBI, and the NCBI accession number is KF040050.

[0025] The surfactant used in the following examples is Tween 80, which may be Span 20 or other surfactants.

[0026] Example 1: Preparation of liquid laccase protective agent

[0027] Formula of liquid laccase protective agent:

[0028] The solvent is NaOH buffer at pH 10.0, including the following components: 2.5% (v / v) trehalose, 3% (v / v) bovine serum albumin, 3% (v / v) glycine, 1.2% (v / v) Tween 80, 1.5% (v / v) sodium alginate, 10% (v / v) glycerol, and 3 mM copper chloride.

[0029] Preparation method: prepare 1mM NaOH buffer, then add trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride into the buffer one by one, and mix evenly to obtain the liquid laccase protective agent.

[0030] Example 2: Preparation of liquid laccase protective agent

[0031] Formula of liquid laccase protective agent:

[0032] The solvent is NaOH buffer at pH 10.0, including the following components: 2% (v / v) trehalose, 2% (v / v) bovine serum albumin, 2% (v / v) glycine, 1% (v / v) Tween 80, 1% (v / v) sodium alginate, 8% (v / v) glycerol, and 2 mM copper chloride.

[0033] Preparation method: prepare 1mM NaOH buffer, then add trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride into the buffer one by one, and mix evenly to obtain the liquid laccase protective agent.

[0034] Example 3: Preparation of liquid laccase protective agent

[0035] Formula of liquid laccase protective agent:

[0036] The solvent is NaOH buffer at pH 10.0, including the following components: 3% (v / v) trehalose, 4% (v / v) bovine serum albumin, 4% (v / v) glycine, 1.4% (v / v) Tween 80, 2% (v / v) sodium alginate, 12% (v / v) glycerol, and 4 mM copper chloride.

[0037] Preparation method: prepare 1mM NaOH buffer, then add trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride into the buffer one by one, and mix evenly to obtain the liquid laccase protective agent.

[0038] Example 4: Preparation of liquid laccase protective agent

[0039] Formula of liquid laccase protective agent:

[0040] The solvent is NaOH buffer at pH 10.0, including the following components: 1.5% (v / v) trehalose, 2% (v / v) bovine serum albumin, 1% (v / v) glycine, 0.8% (v / v) Tween 80, 0.5% (v / v) sodium alginate, 6% (v / v) glycerol, and 1 mM copper chloride.

[0041] Preparation method: prepare 1mM NaOH buffer, then add trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride into the buffer one by one, and mix evenly to obtain the liquid laccase protective agent.

[0042] Example 5: Preparation of liquid laccase protective agent

[0043] Formula of liquid laccase protective agent:

[0044] The solvent is NaOH buffer at pH 10.0, including the following components: 3.5% (v / v) trehalose, 4% (v / v) bovine serum albumin, 5% (v / v) glycine, 1.6% (v / v) Tween 80, 2.5% (v / v) sodium alginate, 14% (v / v) glycerol, and 5 mM copper chloride.

[0045] Preparation method: prepare 1mM NaOH buffer, then add trehalose, bovine serum albumin, glycine, Tween 80, sodium alginate, glycerol and copper chloride into the buffer one by one, and mix evenly to obtain the liquid laccase protective agent.

[0046] Example 6: Comparison of surfactant protection effects

[0047] This example investigates the protective effects of different surfactants on laccase CotA to determine the type of surfactant in the liquid laccase protective agent. The specific steps are as follows:

[0048] Laccase CotA was added to a 1% (v / v) solution of Tween 80, Span 20, Brij 35, NP-40, and Triton X-100 and mixed to obtain a mixture of liquid laccase and Tween 80, Span 20, Brij 35, NP-40, and Triton X-100 solutions, respectively. The mixture was incubated at 70°C (the optimal reaction temperature of laccase CotA) for 3 hours, and the residual enzyme activity was measured to compare the protective effects of different surfactants on laccase CotA. The results are shown in Figure 2. Figure 1 shown.

[0049] from Figure 1 As can be seen, after incubation at 70°C for 3 hours, the Tween 80-treated group had the highest laccase CotA activity retention rate, at 77.9%, followed by the Span 20-treated group, which retained 66.5%. These results indicate that the surfactant Tween 80 is the most effective in protecting CotA laccase, and therefore, Tween 80 was selected as the raw material for the liquid laccase protective agent.

[0050] Example 7: Application of liquid laccase protective agent

[0051] This example investigates the enzyme activity of laccase CotA at 4°C with and without the addition of a liquid laccase protective agent, in order to reflect the effect of the liquid laccase protective agent and the storage stability of laccase. The specific steps are as follows:

[0052] Laccase CotA was added to the liquid laccase protective agent prepared in Example 1 and mixed thoroughly to obtain a mixed solution containing the liquid laccase protective agent. Laccase CotA without the liquid laccase protective agent and the mixed solution were stored at 4°C for a period of time. The changes in the enzyme activity of the liquid laccase before and after storage were measured to monitor changes in the enzyme activity retention rate.

[0053] (1) Determination of laccase CotA activity:

[0054] The protein content was determined using BCA protein quantification.

[0055] Laccase activity was determined by ABTS (ε 420 =3.6×10 4 L·mol -1 cm -1) as the substrate for detection. Prepare 3 mL of the enzyme reaction system. Pipette 2.4 mL of Na2HPO4-acid buffer (0.1 mol / L) and 0.5 mL of the substrate ABTS (final concentration 0.5 mmol / L). Mix evenly and place in a 50°C constant temperature water bath for 5 minutes. Add the diluted laccase enzyme solution and shake to mix thoroughly to start the reaction. Observe the change in absorbance of the reaction system at 420 nm using a spectrophotometer. A reaction using deionized water instead of laccase was used as a control group. Repeat the experiment three times.

[0056] Laccase activity calculation formula: enzyme activity (U / L) = (△OD × V0 × n × 10 6 ) / (△t×V1×ε)

[0057] In the above formula:

[0058] △OD is the difference in absorbance change after the reaction is completed;

[0059] n is the dilution multiple of the enzyme solution;

[0060] V0 is the total volume of the reaction system (mL);

[0061] V1 is the volume of enzyme solution (mL);

[0062] △t is the reaction time (min);

[0063] ε is the molar extinction coefficient, ε 420 =3.6×10 4 L·mol -1 cm -1

[0064] (2) Determination of enzyme activity retention rate:

[0065] Determination of enzyme activity retention at 4°C:

[0066] The laccase CotA with added liquid laccase protective agent was used as the experimental group, and the laccase CotA without added liquid laccase protective agent was used as the control group. The enzyme activity retention rates of the experimental group and the control group laccase CotA were tested at different storage times at 4°C. The test results are shown in Figure 2. Figure 2 shown.

[0067] from Figure 2 It can be seen that after storage at 4°C for different time periods (1 day, 7 days, 14 days, 21 days, 28 days, and 35 days), the laccase CotA activity retention rate in the experimental group was 42.6% at 35 days, an increase of 19.7% compared to the enzyme activity retention rate in the control group. Therefore, the addition of the liquid laccase protective agent of the present invention can significantly improve the storage stability of laccase at 4°C, effectively reduce the loss of enzyme activity of liquid laccase, increase its service life and application effect, and reduce the cost of laccase use.

[0068] In summary, the liquid laccase protective agent of the present invention can achieve long-term storage of laccase, with good enzyme activity and stability, significantly improving the performance of liquid laccase. Compared with laccase without the addition of the protective agent, the enzyme activity of the laccase added after laccase protection increased by 36.1%; the enzyme activity retention rate increased by 19.7% after storage at 4°C for 35 days, improving the storage effect of laccase at 4°C and significantly extending its half-life. The liquid laccase protective agent of the present invention fills the gap in laccase protective agents and facilitates the application of liquid laccase in fields such as food processing, papermaking, textile industry, and industrial wastewater treatment, showing excellent practicality.

[0069] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A liquid laccase protective agent, characterized in that: The liquid laccase protective agent uses NaOH buffer as a solvent and includes the following components: Trehalose, bovine serum albumin, glycine, surfactant, sodium alginate, glycerol and cupric chloride.

2. The liquid laccase protective agent according to claim 1, characterized in that The pH value of the NaOH buffer solution in the liquid laccase protective agent is 10; the concentration of the NaOH buffer solution is 1 mM; The surfactant includes Tween 80 or Span 20.

3. The liquid laccase protective agent according to claim 2, characterized in that The surfactant is Tween 80.

4. The liquid laccase protective agent according to claim 1, characterized in that The liquid laccase protective agent comprises the following components: 1-4% (v / v) trehalose, 1-4% (v / v) bovine serum albumin, 1-7% (v / v) glycine, 0.6-1.8% (v / v) Tween 80, 0.5-2.5% (v / v) sodium alginate, 2-14% (v / v) glycerol, and 1-7 mM copper chloride.

5. The liquid laccase protective agent according to claim 4, characterized in that The liquid laccase protective agent comprises the following components: 2.5% (v / v) trehalose, 3% (v / v) bovine serum albumin, 3% (v / v) glycine, 1.2% (v / v) Tween 80, 1.5% (v / v) sodium alginate, 10% (v / v) glycerol, and 3 mM copper chloride.

6. The liquid laccase protective agent according to claim 1, characterized in that The preparation method of the liquid laccase protective agent comprises: dissolving trehalose, bovine serum albumin, glycine, a surfactant, sodium alginate, glycerol and copper chloride in NaOH buffer one by one to obtain the liquid laccase protective agent.

7. Use of the liquid laccase protective agent according to any one of claims 1 to 6 in improving the storage stability of liquid laccase.

8. The use according to claim 7, characterized in that The liquid laccase is alkaline liquid laccase.

9. The use according to claim 7, characterized in that The liquid laccase is derived from animals, plants or microorganisms.

10. The use according to claim 8, characterized in that The alkaline liquid laccase includes laccase CotA.

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