CN116286731B - Protein-based immobilized enzyme, preparation method thereof and method for efficiently preparing diglyceride - Google Patents

Abstract

The invention discloses a preparation method of protein-based immobilized enzyme, which comprises the following steps: step one, extracting to obtain peanut globulin; adding deionized water to prepare a peanut globulin dispersion liquid, adjusting the pH to be alkaline, and performing acetylation modification to obtain an acetylated peanut globulin solution; step three, the acetylated peanut globulin solution is subjected to heat treatment and salt treatment in sequence to obtain acetylated peanut globulin particles; and fourthly, crosslinking and fixing the acetylated globulin particles and the lipase by using glutaraldehyde, centrifuging to obtain a precipitate after the crosslinking reaction is finished, and adding deionized water for shearing and crushing to obtain the protein-based immobilized enzyme. The invention has the beneficial effects of being applicable to food systems and having high catalysis efficiency of diglycerides. The invention provides a protein-based immobilized enzyme, which has high immobilized efficiency and strong specific activity. Provides a preparation method of diglyceride, which has high preparation efficiency.

Description

Protein-based immobilized enzyme and preparation method thereof and method for efficiently preparing diglyceride

Technical Field

The present invention relates to the technical field of lipase immobilization and application, and more specifically, to a protein-based immobilized enzyme and a preparation method thereof and a method for efficiently preparing diglyceride.

Background technique

Biological enzymes are a common biocatalyst. They can be used in a variety of organic reactions because of their high chemical, regional and stereoselectivity and high catalytic activity under mild reaction conditions. However, when hydrophilic biological enzymes catalyze hydrophobic substrates, the two-phase interface is small, the mass transfer efficiency of the substrate between the two phases is low, and the contact between the enzyme and the substrate is insufficient, resulting in low catalytic efficiency. In order to adapt to organic reaction media and improve recycling, emulsification is currently commonly used to increase the reaction contact area and improve catalytic efficiency. In order to maintain the stability of the emulsion and the catalytic interface, it is usually necessary to add small molecule surfactants or violent stirring. However, small molecule surfactants are difficult to separate from the product, which will bring safety issues. In addition, small molecule surfactants and violent stirring will destroy the enzyme structure to varying degrees, thereby affecting enzyme activity.

In Example 1 of the Chinese application with application number 201810430317.7, a method for preparing NADPH by fixing GDH using silica gel as a carrier is disclosed. When silica gel is used as a carrier of biological enzymes, silica gel has low degradability and certain biological toxicity, which limits its application in the food field. However, the present invention uses natural peanut globulin materials to obtain protein-based lipase solid particles with high safety and no problems such as biodegradability.

The Chinese application, with application number 202111552631.0, discloses in Example 1 a method of preparing an immobilized enzyme using paraffin, chitosan, polyacrylamide, and monostearyl glyceride under the action of a high-speed homogenizer, and encapsulating the lipase in a core-shell structure of paraffin microspheres. This method introduces materials such as polyacrylamide and paraffin, which limits its application in the fields of biomedicine and food. However, the present invention does not introduce such substances, which broadens its application in food catalysis.

Pickering emulsions have been proven to be useful for improving catalytic efficiency. This system avoids the addition of small molecule surfactants and high-energy emulsification is not necessary. At present, inorganic particles, inorganic polymer particles, etc. are commonly used to cross-link and fix biological enzymes to construct Pickering emulsion catalytic systems. However, these particles have low biocompatibility and prominent safety issues. It can be seen that it is necessary to prepare food-grade particles based on natural macromolecules for fixing biological enzymes to construct Pickering emulsion catalytic systems. In view of this, the present invention is specially proposed.

Summary of the invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

In order to achieve these purposes and other advantages according to the present invention, a method for preparing a protein-based immobilized enzyme is provided, comprising the following steps:

Step 1, extracting and obtaining peanut globulin;

Step 2: adding deionized water to peanut globulin to prepare a peanut globulin dispersion, adjusting the pH to alkaline, and performing acetylation modification to obtain an acetylated peanut globulin solution;

Step 3, the acetylated peanut globulin solution is subjected to heat treatment and salt treatment in sequence to obtain acetylated peanut globulin particles;

Step 4: Use glutaraldehyde to cross-link and fix the acetylated globulin particles and lipase. After the cross-linking reaction is completed, centrifuge to obtain the precipitate, add deionized water to shear and crush it, and obtain the protein-based immobilized enzyme.

Preferably, the method for extracting peanut globulin is as follows: peanut defatted powder is mixed with a phosphate buffer solution having a pH value of 6.9 to 7.5 and a concentration of 0.35 to 0.45 mol/L at a solid-liquid ratio of 1 g:1.5 to 3.5 mL, centrifuged at 6000 to 10000 rpm for 20 to 40 min, the precipitate is removed, the supernatant is cooled and precipitated at 2 to 5° C. for a certain period of time, centrifuged at 2 to 5° C. and 6000 to 10000 rpm for 20 to 40 min, the precipitate is taken, and freeze-dried or spray-dried to obtain peanut globulin.

Preferably, the acetylation modification method is as follows: adjusting the pH value of the peanut globulin dispersion to 9.5-11, the content of peanut globulin in the peanut globulin dispersion to 6.5wt%, adding 0-50wt% succinic anhydride of peanut globulin, stirring for 20-50min, and maintaining the pH value at 9.5-11, dialyzing at 2-4°C using a 10kDa dialysis bag for 24-48h to obtain an acetylated peanut globulin solution.

Preferably, the heat treatment in step three is specifically: water bath at 85-95° C. for 15-45 min.

Preferably, the salt treatment in step 3 is specifically as follows: adjusting the pH to 5-6.5 with hydrochloric acid, and then adding NaCl particles to a concentration of 500-900 mmol to obtain acetylated peanut globulin particles.

Preferably, in step 4, the concentration of peanut globulin in the acetylated peanut globulin solution is 2.0-7.0wt%, the concentration of the glutaraldehyde reagent is 0-3.0%, the concentration of the lipase solution is 5.0-15.0%, the cross-linking time is 0.5-4.0h, the volume ratio of the lipase solution to the acetylated peanut globulin solution is 1:3-7, and the volume ratio of the glutaraldehyde reagent to the acetylated peanut globulin solution is 20-30:1.

Preferably, after the cross-linking is completed, the centrifugation condition is 120,000 rpm for 15 min, the supernatant is removed, the precipitate is taken, deionized water is added to the volume before centrifugation, and shearing and crushing is performed at 6,000 rpm for 1 min.

Preferably, the lipase is Lipozyme TL 100L.

Provided is a protein-based immobilized enzyme prepared by the above preparation method.

A method for preparing diglyceride based on the above protein-based immobilized enzyme is provided, comprising the following steps:

Step a, adding protein-based immobilized enzyme into oil phase to prepare Pickering emulsion;

Step b: reacting the Pickering emulsion at 45° C. for a certain period of time to obtain diglyceride.

The present invention has at least the following beneficial effects:

First, the present invention cross-links and fixes lipase and acetylated peanut globulin, and the obtained protein-based immobilized enzyme particles have higher immobilization efficiency and specific activity after optimization, and can stabilize W/O type Pickering emulsion to construct a Pickering emulsion catalytic system.

Second, the catalytic system constructed by the present invention can effectively improve the efficiency of catalytic preparation of diglyceride oil, and create a new approach and method for preparing diglyceride oil.

Third, the method for preparing the protein-based immobilized enzyme provided by the present invention is simple to operate, safe to use, and has improved catalytic efficiency, which is conducive to its implementation in production in the food industry.

Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph showing specific activity test data of the present invention;

Fig. 2 is a diglyceride content curve of the present invention;

FIG3 is a diagram showing the dispersion state of peanut globulin and acetylated peanut globulin of the present invention at pH 5.5;

FIG. 4 shows the hydrophobicity of acetylated arachidone without hot salt treatment and with hot salt treatment according to the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

<Example 1>

Provided is a protein-based immobilized lipase particle, the preparation method of which comprises the following steps:

Step 1, weigh a certain mass of peanut globulin, add it into a certain volume of deionized water while stirring, to prepare a peanut protein dispersion, and adjust its pH value to 10.5, then add 30% of the dry weight of peanut globulin succinic anhydride, keep the pH value at 10.5, stir for 30 minutes, and then dialyze at 4°C using a 10kDa dialysis bag for 24 hours to obtain an acetylated peanut globulin solution;

The acetylated peanut globulin solution was placed in a constant temperature water bath at 90°C for 30 min;

The pH value of the acetylated arachidone solution was adjusted to 5.5 with 1 M hydrochloric acid, and 700 mmol of NaCl particles were added to obtain acetylated arachidone particles.

Step 2, the concentration of acetylated peanut globulin particles is 6.5% (based on the mass of dry peanut globulin), the concentration of glutaraldehyde reagent is 0.5%, the amount of glutaraldehyde reagent added is 400 μL, the concentration of Lipozyme TL (lipase solution) 100L is 10.0% (the concentration of lipase prepared using Tris-HC buffer), the ratio of lipase solution to acetylated peanut globulin particles is 1:4 (2mL:8mL), the acetylated peanut globulin particles, glutaraldehyde reagent, and lipase solution are mixed, and the cross-linking reaction is stirred at 300 rpm at room temperature for 1.0 h;

After the cross-linking reaction was completed, the reaction mixture was centrifuged at 120,000 rpm for 15 min, the supernatant was removed, the precipitate was taken, deionized water was added to the volume before centrifugation, and sheared and crushed at 6,000 rpm for 1 min to obtain a protein-based immobilized enzyme solution.

Step 3, using the protein-based immobilized enzyme solution prepared in step 2 to prepare a Pickering emulsion, the specific shearing method is: soybean oil is first sheared at 10000 rpm for 1 minute, then the protein-based immobilized enzyme solution is added, and then sheared for another 1 minute to obtain a Pickering emulsion; wherein the mass ratio of soybean oil to protein-based immobilized enzyme solution is 4:1.

The Pickering emulsion was reacted at 45°C for 0 to 240 min, and the diglyceride content was determined.

The immobilization efficiency and specific activity of the protein-based immobilized lipase particles in this example are shown in Table 1, and the specific activity is shown in the darker single column in FIG1 .

Table 1 Immobilization efficiency and specific activity of protein-based immobilized lipase particles

project Solidification efficiency % Specific activity U/mg Example 1 Protein-based immobilized lipase particles 7.17±0.006 1.06±0.082

<Example 2>

Provided are a preparation method and catalytic application of protein-based immobilized lipase particles, the preparation method comprising the following steps:

Step 1: Prepare acetylated peanut globulin particles in the same manner as in Example 1.

Step 2, the concentration of acetylated peanut globulin particles is 6.5% (based on the mass of dry peanut globulin), the concentration of glutaraldehyde reagent is 1.0%, the amount of glutaraldehyde reagent added is 400 μL, the concentration of Lipozyme TL (lipase solution) 100L is 10.0%, the ratio of lipase solution to acetylated peanut globulin particles is 1:4 (2mL:8mL), the acetylated peanut globulin particles, glutaraldehyde reagent, and lipase solution are mixed, and the cross-linking reaction is stirred at 300 rpm at room temperature for 1.0h;

After the cross-linking was completed, the reaction mixture was centrifuged at 120,000 rpm for 15 min, the supernatant was removed, the precipitate was taken, deionized water was added to the volume before centrifugation, and sheared and crushed at 6,000 rpm for 1 min to obtain a protein-based immobilized enzyme solution.

Step 3: Prepare a Pickering emulsion using the protein-based immobilized enzyme solution prepared in step 2. The specific shearing method is: first shear the soybean oil at 10,000 rpm for 1 minute, then add the protein-based immobilized enzyme solution and shear for another 1 minute. The obtained Pickering emulsion has a mass ratio of soybean oil to protein-based immobilized enzyme solution of 4:1.

The Pickering emulsion was reacted at 45°C for 0 to 240 min, and the diglyceride content was determined.

The immobilization efficiency and specific activity of the protein-based immobilized lipase particles in this example are shown in Table 2, and the specific activity is shown in the dark single column in Figure 1. The diglyceride content curve in this example is shown in the dark dot curve in Figure 2.

Table 2 Immobilization efficiency and specific activity of protein-based immobilized lipase particles

project Solidification efficiency % Specific activity U/mg Example 2 Protein-based immobilized lipase particles 7.20±0.003 1.47±0.153

<Comparative Example 1>

A method for preparing diglyceride by catalysis of free lipase is provided, comprising the following steps:

Step 1: Prepare 0.26% Lipozyme TL 100L (lipase solution).

Step 2: Use the above-mentioned free lipase solution and mix with soybean oil for shearing to prepare a two-phase catalytic reaction system. The shearing method is to shear the soybean oil at 10,000 rpm for 1 minute, add the free lipase solution and then shear for another 1 minute to obtain a two-phase system;

The two-phase system was reacted at 45°C for 0 to 240 min, and the diglyceride content was determined.

The specific activity of free lipase in this comparative example is shown in the light-colored single column in FIG1 , and the diacylglycerol content curve is shown in the light-colored square curve in FIG2 .

<Comparative Example 2>

After acetylation, the amino group of peanut globulin is combined with the acyl group, the positive charge is eliminated, the electron cloud density increases, the negative charge of the molecule increases, and the isoelectric point shifts to the left. Under this pH condition, the modified particles are negatively charged and repel each other without aggregation and precipitation. Therefore, the peanut globulin can still maintain a uniform dispersion state at pH 5.5 without sedimentation, which is beneficial to the subsequent lipase immobilization and emulsion stabilization.

The dispersion state of unacetylated peanut globulin at pH 5.5 is shown in Figure 3. At the same time, the surface hydrophobicity (Ho) of the acetylated protein is improved (as shown in Figure 4), which is more inclined to stabilize the W/O Pickering emulsion.

<Comparative Example 3>

The other steps are the same as those in Example 1, except that no heat treatment or salt treatment is performed.

In the experiment, the hot salt method was used to prepare acetylated peanut globulin particles. After heat treatment, the hydrophobic groups in the acetylated peanut globulin molecules were exposed, the hydrophobic effect of the protein was enhanced, and Ho was significantly improved (as shown in Figure 4); at the same time, after salt treatment, due to the electrostatic shielding effect, the electrostatic repulsion between protein molecules was reduced, and the protein underwent slight "salting out", resulting in acetylated peanut globulin particles.

Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims (8)

1. A method for preparing a protein-based immobilized enzyme for preparing diglyceride, characterized in that it comprises the following steps: Step 1, extracting and obtaining peanut globulin; Step 2: adding deionized water to peanut globulin to prepare a peanut globulin dispersion, adjusting the pH to alkaline, and performing acetylation modification to obtain an acetylated peanut globulin solution; Step 3, the acetylated peanut globulin solution is subjected to heat treatment and salt treatment in sequence to obtain acetylated peanut globulin particles; Step 4: Use glutaraldehyde to cross-link and fix the acetylated globulin particles and lipase. After the cross-linking reaction is completed, centrifuge to obtain the precipitate, add deionized water to shear and crush it, and obtain the protein-based immobilized enzyme; The specific method for extracting peanut globulin is as follows: peanut defatted powder is mixed with a phosphate buffer solution having a pH value of 6.9 to 7.5 and a concentration of 0.35 to 0.45 mol/L at a solid-liquid ratio of 1 g:1.5 to 3.5 mL, centrifuged at 6000 to 10000 rpm for 20 to 40 min, the precipitate is removed, the supernatant is cooled and precipitated at 2 to 5° C. for a certain period of time, centrifuged at 2 to 5° C. and 6000 to 10000 rpm for 20 to 40 min, the precipitate is taken, and freeze-dried or spray-dried to obtain peanut globulin; The acetylation modification method is specifically as follows: the pH value of the peanut globulin dispersion is adjusted to 9.5-11, the content of peanut globulin in the peanut globulin dispersion is 6.5wt%, 0-50wt% of succinic anhydride of peanut globulin is added, stirred for 20-50min, and the pH value is maintained at 9.5-11, dialyzed at 2-4°C using a 10kDa dialysis bag for 24-48h to obtain an acetylated peanut globulin solution. 2. The method for preparing a protein-based immobilized enzyme for preparing diglyceride according to claim 1, characterized in that the heat treatment in step 3 is specifically: water bathing at 85-95°C for 15-45 minutes. 3. The method for preparing a protein-based immobilized enzyme for preparing diglyceride according to claim 1, characterized in that the salt treatment in step 3 is specifically: adjusting the pH to 5-6.5 with hydrochloric acid, and then adding NaCl particles to a NaCl concentration of 500-900 mmol to obtain acetylated peanut globulin particles. 4. The method for preparing a protein-based immobilized enzyme for preparing diglyceride according to claim 1, characterized in that the concentration of peanut globulin in the acetylated peanut globulin solution in step 4 is 2.0-7.0wt%, the concentration of the glutaraldehyde reagent is 0-3.0%, the concentration of the lipase solution is 5.0-15.0%, the cross-linking time is 0.5-4.0h, the volume ratio of the lipase solution to the acetylated peanut globulin solution is 1:3-7, and the volume ratio of the glutaraldehyde reagent to the acetylated peanut globulin solution is 20-30:1. 5. The method for preparing a protein-based immobilized enzyme for preparing diglyceride according to claim 1, characterized in that after the cross-linking is completed, the centrifugation condition is 120,000 rpm, centrifugation is performed for 15 minutes, the supernatant is removed, the precipitate is taken, deionized water is added to the volume before centrifugation, and shearing and crushing is performed at 6,000 rpm for 1 minute. 6. The method for preparing a protein-based immobilized enzyme for preparing diglyceride according to claim 1, wherein the lipase is Lipozyme TL 100L. 7. The protein-based immobilized enzyme for preparing diacylglycerol obtained by the preparation method according to any one of claims 1 to 6. 8. A method for preparing diglyceride based on the protein-based immobilized enzyme for preparing diglyceride according to claim 7, characterized in that it comprises the following steps: Step a, adding protein-based immobilized enzyme into oil phase to prepare Pickering emulsion; Step b: reacting the Pickering emulsion at 45° C. for a certain period of time to obtain diglyceride.