Liquid beta-1, 4 endo-xylanase protective agent and application thereof

Abstract

The invention provides a liquid beta-1, 4 endo-xylanase protective agent and application thereof, and belongs to the technical field of enzyme engineering. The liquid beta-1, 4 endo-xylanase protective agent provided by the invention takes a PBS (Phosphate Buffer Solution) with the pH value of 7.8 as a solvent, and comprises methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine and manganese chloride; the liquid beta-1, 4 endo-xylanase protective agent can effectively reduce the enzyme activity loss of beta-1, 4 endo-xylanase in the storage and transportation processes and improve the storage stability of the liquid beta-1, 4 endo-xylanase, so that the service life of the liquid beta-1, 4 endo-xylanase is prolonged, the application effect of the liquid beta-1, 4 endo-xylanase is improved, the use cost of the liquid beta-1, 4 endo-xylanase is reduced, and the liquid beta-1, 4 endo-xylanase protective agent is beneficial for improving the stability of the liquid beta-1, 4 endo-xylanase. The endo-xylanase disclosed by the invention is applied to the fields of feed processing, food processing, pulping processing, industrial wastewater treatment and the like, and has good practicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a liquid β-1,4-endoxylanase protectant and its application. Background Art

[0002] β-1,4-endoxylanase is an industrially valuable enzyme. According to the difference in the pH range when it acts on the substrate, it can be divided into acidic β-1,4-endoxylanase and alkaline β-1,4-endoxylanase. Compared with acidic β-1,4-endoxylanase, alkaline β-1,4-endoxylanase has a broad market prospect and great development potential, and is mainly applied to the brewing, feed processing, and pulp and paper industries, etc.

[0003] β-1,4-endoxylanase can also be divided into solid β-1,4-endoxylanase and liquid β-1,4-endoxylanase. Since solid β-1,4-endoxylanase is easy to store, transport, and is beneficial to protecting the activity of β-1,4-endoxylanase, solid β-1,4-endoxylanase is mainly on the market at present. However, solid β-1,4-endoxylanase has defects such as complex production technology, high production cost, and narrow application range, and cannot be widely applied. In contrast, the production process of liquid β-1,4-endoxylanase is simple, the production cost is low, and it has a wider application range. However, the enzyme activity of liquid β-1,4-endoxylanase will rapidly decline during storage and transportation, seriously affecting its application effect. Therefore, a protectant is needed to reduce the loss of enzyme activity of liquid β-1,4-endoxylanase during storage and transportation and improve its service life. Summary of the Invention

[0004] In view of some deficiencies in the prior art, the present invention provides a liquid β-1,4-endoxylanase protectant and its application; the liquid β-1,4-endoxylanase protectant of the present invention uses PBS buffer solution with a pH of 7.8 as the solvent, and includes: methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride; the liquid β-1,4-endoxylanase protectant can effectively reduce the loss of enzyme activity of β-1,4-endoxylanase during storage and transportation, improve the storage stability of liquid β-1,4-endoxylanase, thereby improving its service life and application effect, reducing the use cost of β-1,4-endoxylanase, and contributing to the application of liquid β-1,4-endoxylanase in the fields of feed processing, food processing, pulp processing, and industrial wastewater treatment, etc., and has good practicability.

[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 β-1,4-endoxylanase protectant, which uses PBS buffer as a solvent and includes the following components:

[0007] methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride.

[0008] Preferably, the pH value of the PBS buffer as the solvent in the liquid β-1,4-endoxylanase protectant is 7.8.

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

[0010] Preferably, the liquid β-1,4-endoxylanase protectant contains the following components: 1-8% (v / v) methanol, 1-8% (v / v) glycerol, 1-9% (v / v) gelatin, 1-9% (v / v) polyethylene glycol, 1-8% (v / v) trehalose, 1-8% (v / v) glycine, 20 mM-160 mM manganese chloride.

[0011] Preferably, the liquid β-1,4-endoxylanase protectant contains the following components: 3.735% (v / v) methanol, 3% (v / v) glycerol, 0.768% (v / v) gelatin, 3% (v / v) polyethylene glycol, 5% (v / v) trehalose, 6% (v / v) glycine, 58.380 mM manganese chloride.

[0012] Preferably, the preparation method of the liquid β-1,4-endoxylanase protectant includes: dissolving methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride into the PBS buffer one by one to obtain the liquid β-1,4-endoxylanase protectant.

[0013] The present invention also provides the application of the above liquid β-1,4-endoxylanase protectant in improving the storage stability of liquid β-1,4-endoxylanase.

[0014] Preferably, the liquid β-1,4-endoxylanase is an alkaline liquid β-1,4-endoxylanase.

[0015] Preferably, the liquid β-1,4-endoxylanase is derived from animals, plants, or microorganisms.

[0016] Preferably, the alkaline liquid β-1,4-endoxylanase includes β-1,4-endoxylanase XynH; the amino acid sequence of the β-1,4-endoxylanase XynH is shown in SEQ ID NO.1, and the nucleotide sequence encoding the β-1,4-endoxylanase XynH is shown in SEQ ID NO.2.

[0017] Preferably, the application includes: mixing a liquid β-1,4-endoxylanase protectant and a liquid β-1,4-endoxylanase in a volume ratio of 2:1 for use.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention uses PBS buffer solution with a pH of 7.8 as a solvent, and adds methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine and manganese chloride thereto to prepare the liquid β-1,4-endoxylanase protectant. In the liquid β-1,4-endoxylanase protectant, the network structure formed by thickeners such as gelatin can produce a sieve effect, spatially limit the smaller enzyme protein molecules, reduce the mutual collision of enzyme molecules, and improve the thermal stability of enzyme molecules to a certain extent; trehalose can maintain the structural stability of β-1,4-endoxylanase, stable osmotic volume, and can resist oxidative damage; alcohols such as glycerol, polyethylene glycol, and methanol have a certain effect on the stability of the enzyme. The stability mechanism may be the interaction between the hydroxyl group and the enzyme molecule, or the alcohols reduce the dielectric constant of the medium, strengthen the hydrophobic interaction of the enzyme molecule, these substances combine with water molecules, reduce the water activity, and reduce the influence of water molecules on the enzyme protein; glycine amino acid ions have amphoteric acid-base properties, so they can inhibit the pH change of the solution during the low-temperature preservation and freeze-drying process of biological products, protect the active centers of glutamine, threonine and asparagine of the alkaline liquid β-1,4-endoxylanase, so as to achieve the purpose of protecting the active components; manganese chloride not only has a protective effect, but also has an activating effect. It can not only protect the structural stability of β-1,4-endoxylanase, but also play a bridging role between β-1,4-endoxylanase and the substrate to form a complex, which is beneficial to the binding of the substrate to the essential groups of the active center of β-1,4-endoxylanase, and thus improves the enzyme activity.

[0020] In the liquid β-1,4-endoxylanase protectant of the present invention, trehalose, polyethylene glycol and manganese chloride can react synergistically with the alkaline liquid β-1,4-endoxylanase to form a polyhydroxy hydrogen bond structure, which stabilizes the secondary structure of β-1,4-endoxylanase, further increases the stability of β-1,4-endoxylanase, and can replace water molecules to maintain the spatial structure stability of the enzyme.

[0021] In addition, through experimental verification of the storage stability, the liquid β-1,4-endoxylanase protectant of the present invention can effectively reduce the loss of enzyme activity of the liquid β-1,4-endoxylanase, improve its service life and application effect, and reduce the use cost of the β-1,4-endoxylanase. Compared with the enzyme without the protectant, the enzyme activity retention rates can be increased by 41.2%, 27.6% and 8.9% respectively when stored at room temperature, 4°C and -80°C for 30 days. The liquid β-1,4-endoxylanase protectant of the present invention fills the gap of the β-1,4-endoxylanase protectant and has good practicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the standard curve of β-1,4-endoxylanase XynH.

[0023] Figure 2 It is the influence of the liquid β-1,4-endoxylanase protectant on the storage stability of alkaline β-1,4-endoxylanase under room temperature conditions.

[0024] Figure 3 It is the influence of the liquid β-1,4-endoxylanase protectant on the storage stability of alkaline β-1,4-endoxylanase under 4°C conditions.

[0025] Figure 4 It is the influence of the liquid β-1,4-endoxylanase protectant on the storage stability of alkaline β-1,4-endoxylanase under -80°C conditions. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be further described below in conjunction with the drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. In the following embodiments, various processes and methods not described in detail are all conventional methods well known in the art. The sources, trade names of the reagents used, and those for which it is necessary to list their components are indicated when they first appear. For the same reagents used later, without special instructions, they are the same as those indicated in the first appearance; the reagents, materials, etc. involved, without special instructions, are obtained for commercial use.

[0027] The liquid β-1,4-endoxylanase described in the present invention is an alkaline β-1,4-endoxylanase; the liquid β-1,4-endoxylanase can be derived from animals, plants or microorganisms. The following takes β-1,4-endoxylanase XynH as an example to verify the effect of the liquid β-1,4-endoxylanase protectant. The amino acid sequence of the β-1,4-endoxylanase is shown in SEQ ID NO.1, and the nucleotide sequence encoding β-1,4-endoxylanase XynH is shown in SEQ ID NO.2.

[0028] SEQ ID NO.1:

[0029] MKILPSVLILLLGCVPVFSSQNVSLRELAEKLNIYIGFAAINNFWSLSDAEKYMEVARREFNILTPENQMKWDTIHPERDRYNFTPAEKHVEFAEENDMIVHGHTLVWHNQLPGWITGREWTKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDSGTYRESVWYKTIGPEYIEKAFRWAKEADPDAILIYNDYSIEEINAKSNFVYNMIKELKEKGVPVDGIGFQMHIDYRGLNYDSFRRNLERFAKLGLQIYITEMDVRIPLSGSEEYYLKKQAEVCAKIFDICLDNPAVKAIQFWGFTDKYSWVPGFFKGYGKALLFDENYNPKPCYYAIKEVLEKKIEERK*

[0030] SEQ ID NO.2:

[0031]

[0032] In the following examples, the method for measuring the enzyme activity of xylan is as follows:

[0033] ① Draw the standard curve of xylan:

[0034] Measure the absorbance values of xylose at different concentrations at 480 nm respectively, and draw the standard curve of xylan. The results are as Figure 1 shown. It can be seen from Figure 1 that the absorbances of aqueous xylose solutions with concentrations of 3 μmol / ml, 4 μmol / ml, 6 μmol / ml, 7 μmol / ml, and 8 μmol / ml at 480 nm are positively correlated, and the linear equation is: y = 0.1019x - 0.1167, R 2 = 0.9933.

[0035] ② Experimental group: Accurately measure 1.00 mL of the enzyme solution of β-1,4-endoxylanase into a 25-mL colorimetric tube, place it in a water bath shaker at different temperatures required for the investigation experiment and with a shaking speed of 100 times / min for preheating for 10 min. Add 2.00 mL of beech xylan solution preheated at 50 °C, place it in the water bath shaker for incubation for 10 min (accurately time), and immediately add 2.5 mL of DNS reagent to terminate the reaction. Then heat the colorimetric tube in a boiling water bath for 3 min (accurately time), cool it to room temperature with water, and add distilled water to 25 mL to obtain the sample test solution.

[0036] Blank group: Heat the β-1,4-endoxylanase in a boiling water bath for 3 min (accurately time), and then perform the above operations after cooling it to room temperature with water.

[0037] Measure the xylan production contents in the experimental group and the blank group respectively through the standard curve prepared in ①.

[0038] The definition of the specific activity of β-1,4-endoxylanase: The β-1,4-endoxylanase required to produce 1 μmol of xylan in 1 min, and the unit of specific activity: (U / mg). The experiment is set with three replicates.

[0039] Example 1: Preparation of liquid β-1,4-endoxylanase protectant

[0040] The formula of the liquid β-1,4-endoxylanase protectant, where the solvent is PBS buffer (pH 7.8):

[0041]

[0042] Preparation method:

[0043] Methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride were added one by one to PBS buffer, and mixed evenly to obtain the liquid β-1,4-endoxylanase protectant.

[0044] Example 2: Preparation of Liquid β-1,4-Endoxylanase Protectant

[0045] Formulation of the liquid β-1,4-endoxylanase protectant, where the solvent is PBS buffer (pH 7.8):

[0046]

[0047]

[0048] Preparation method:

[0049] Methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride were added one by one to PBS buffer, and mixed evenly to obtain the liquid β-1,4-endoxylanase protectant.

[0050] Example 3: Preparation of Liquid β-1,4-Endoxylanase Protectant

[0051] Formulation of the liquid β-1,4-endoxylanase protectant, where the solvent is PBS buffer (pH 7.8):

[0052]

[0053] Preparation method:

[0054] Methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride were added one by one to PBS buffer, and mixed evenly to obtain the liquid β-1,4-endoxylanase protectant.

[0055] Example 4: Preparation of Liquid β-1,4-Endoxylanase Protectant

[0056] Formulation of the liquid β-1,4-endoxylanase protectant, where the solvent is PBS buffer (pH 7.8):

[0057]

[0058] Preparation method:

[0059] Methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine, and manganese chloride were added one by one to PBS buffer, and mixed evenly to obtain the liquid β-1,4-endoxylanase protectant.

[0060] Example 5: Application of Liquid β-1,4-Endoxylanase Protectant

[0061] In this example, taking the liquid β-1,4-endoxylanase protectant prepared in Example 4 as an example, the enzyme activity of β-1,4-endoxylanase XynH with or without the liquid β-1,4-endoxylanase protectant was investigated under different environments to evaluate the improvement effect of the liquid β-1,4-endoxylanase protectant on the storage stability of β-1,4-endoxylanase. The specific steps are as follows:

[0062] Experimental group: β-1,4-endoxylanase XynH was added to the liquid β-1,4-endoxylanase protectant prepared in Example 1 and mixed evenly to obtain a mixed solution containing the liquid β-1,4-endoxylanase protectant.

[0063] Control group: β-1,4-endoxylanase XynH without the liquid β-1,4-endoxylanase protectant.

[0064] The control group and the experimental group were stored at room temperature, 4°C, and -80°C for a period of time respectively. The changes in enzyme activity of the liquid β-1,4-endoxylanase before and after storage were measured, and the changes in its enzyme activity retention rate were monitored. Among them, the enzyme activity retention rate (%) = (residual enzyme activity / initial enzyme activity) × 100%.

[0065] From Figure 2 It can be seen that when stored at room temperature for different times (1d, 2d, 3d, 4d, 5d, 15d, 30d), until 30d, the enzyme activity retention rate of β-1,4-endoxylanase XynH in the experimental group was 85.49%, which was 41.2% higher than that in the control group. It can be seen that adding the liquid β-1,4-endoxylanase protectant of the present invention can significantly improve the storage stability of β-1,4-endoxylanase at room temperature, effectively reduce the enzyme activity loss of the liquid β-1,4-endoxylanase, improve its service life and application effect, and reduce the use cost of β-1,4-endoxylanase.

[0066] From Figure 3 It can be seen that when stored at 4°C for different times (1d, 2d, 3d, 4d, 5d, 15d, 30d), until 30d, the enzyme activity retention rate of β-1,4-endoxylanase XynH in the experimental group was 81.34%, which was 27.6% higher than that in the control group. It can be seen that adding the liquid β-1,4-endoxylanase protectant of the present invention can significantly improve the storage stability of β-1,4-endoxylanase at 4°C, effectively reduce the enzyme activity loss of the liquid β-1,4-endoxylanase, improve its service life and application effect, and reduce the use cost of β-1,4-endoxylanase.

[0067] From Figure 4It can be seen that when stored at -80°C for different times (1d, 2d, 3d, 4d, 5d, 15d, 30d), until 30d, the enzyme activity retention rate of β-1,4-endoxylanase XynH in the experimental group was 23.10%, which was 8.9% higher than that in the control group. It can be seen that adding the liquid β-1,4-endoxylanase protectant of the present invention can significantly improve the storage stability of β-1,4-endoxylanase at -80°C, effectively reduce the enzyme activity loss of the liquid β-1,4-endoxylanase, improve its service life and application effect, and reduce the use cost of β-1,4-endoxylanase.

[0068] In summary, the liquid β-1,4-endoxylanase protectant of the present invention can achieve the long-term preservation of β-1,4-endoxylanase, with good enzyme activity and stability, and significantly improve the performance of the liquid β-1,4-endoxylanase. The enzyme activity retention rates of β-1,4-endoxylanase added with the liquid β-1,4-endoxylanase protectant are increased by 41.2%, 27.6% and 8.9% respectively when stored at room temperature, 4°C and -80°C for 30d compared with those without addition. The liquid β-1,4-endoxylanase protectant can effectively reduce the enzyme activity loss of the liquid β-1,4-endoxylanase, improve the service life and application effect of β-1,4-endoxylanase, reduce the use cost of β-1,4-endoxylanase, and contribute to the application of the liquid β-1,4-endoxylanase in the fields of feed processing, food processing, pulp processing and industrial wastewater treatment.

[0069] The above embodiments are the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Without departing from the essential content of the present invention, any obvious improvements, substitutions or variations that those skilled in the art can make all belong to the protection scope of the present invention.

Claims

1. A liquid β-1,4-endo-xylanase protective agent, characterized in that: The liquid β-1,4 endo-xylanase protective agent uses PBS buffer as a solvent and includes the following components: Methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine and manganese chloride.

2. The liquid β-1,4-endo-xylanase protective agent according to claim 1, characterized in that: The pH value of the solvent PBS buffer in the liquid β-1,4 endo-xylanase protecting agent is 7.8; the concentration of the PBS buffer is 1 mM.

3. The liquid β-1,4-endo-xylanase protecting agent according to claim 1, characterized in that: The liquid β-1,4 endo-xylanase protective agent comprises the following components: 1-8% (v / v) methanol, 1-8% (v / v) glycerol, 1-9% (v / v) gelatin, 1-9% (v / v) polyethylene glycol, 1-8% (v / v) trehalose, 1-8% (v / v) glycine, and 20mM-160mM manganese chloride.

4. The liquid β-1,4-endo-xylanase protecting agent according to claim 3, characterized in that: The liquid β-1,4 endo-xylanase protective agent comprises the following components: 3.735% (v / v) methanol, 3% (v / v) glycerol, 0.768% (v / v) gelatin, 3% (v / v) polyethylene glycol, 5% (v / v) trehalose, 6% (v / v) glycine, and 58.380 mM manganese chloride.

5. The liquid β-1,4-endo-xylanase protecting agent according to claim 1, characterized in that: The preparation method of the liquid β-1,4 endo-xylanase protective agent comprises: dissolving methanol, glycerol, gelatin, polyethylene glycol, trehalose, glycine and manganese chloride into PBS buffer one by one to obtain the liquid β-1,4 endo-xylanase protective agent.

6. Use of the liquid β-1,4-endo-xylanase protective agent according to any one of claims 1 to 5 in improving the storage stability of liquid β-1,4-endo-xylanase.

7. The use according to claim 6, characterized in that: The liquid β-1,4 endo-xylanase is derived from animals, plants or microorganisms.

8. The use according to claim 6, characterized in that: The liquid β-1,4 endo-xylanase is an alkaline liquid β-1,4 endo-xylanase.

9. The use according to claim 8, characterized in that: The alkaline liquid β-1,4 endo-xylanase comprises ββ-1,4 endo-xylanase XynH; the amino acid sequence of the β-1,4 endo-xylanase XynH is shown in SEQ ID NO.1, and the nucleotide sequence encoding the β-1,4 endo-xylanase XynH is shown in SEQ ID NO.

2.

10. The use according to claim 8, characterized in that: The application comprises: mixing the liquid β-1,4 endo-xylanase protective agent and the liquid β-1,4 endo-xylanase in a volume ratio of 2:1.

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