Preparation method of high-heat-resistance ferritin

Abstract

The invention relates to a preparation method of high-heat-resistance ferritin, and relates to the field of food processing. A salt bridge network structure is added on a ferritin C3 interface, so that the thermal denaturation temperature of the ferritin is remarkably improved. In the aspect of application, due to the high thermal stability of the mutant ferritin, the mutant ferritin has a more remarkable protection effect on the loaded functional factors at high temperature, which is of great significance to the application of the functional factors in the food processing industry.

Description

Technical Field

[0001] The present invention belongs to the field of food processing, and particularly relates to a method for preparing highly heat-resistant ferritin. Background Art

[0002] Ferritin is widely distributed in various organisms such as microorganisms, animals and plants, and its higher-level structure is relatively conserved; ferritin is composed of 24 subunits and self-assembles into a hollow spherical structure with inner and outer diameters of about 8 nm and 12 nm respectively. Its unique natural nano-cavity structure has attracted extensive attention in the aspect of multifunctional nano-carriers. First, the natural nano-cavity structure provides an excellent basis for encapsulating and delivering bioactive small molecules; second, the tight outer shell structure of ferritin can effectively prevent the leakage of the encapsulated contents, thereby ensuring the loading efficiency of the encapsulated substances; finally, the water solubility, stability, etc. of small molecules loaded in the inner cavity of ferritin, such as curcumin, quercetin, etc., have also been greatly improved. Generally, ferritin is dissociated into subunits under the conditions of pH ≤ 2 or ≥ 12, but when the pH is restored to neutral, the ferritin subunits reassemble to form a cage-like structure.

[0003] Although ferritin nanocages show relatively good thermal stability compared with other biomaterials, it is still inevitable to face higher-temperature heat treatment (such as food processing, etc.) during application. Therefore, improving the heat tolerance of ferritin is of great significance for broadening the application of ferritin. Summary of the Invention

[0004] The present invention provides a method for preparing highly heat-resistant ferritin and its application in improving the thermal stability of small molecules. By modifying ferritin at the C 3 interface to increase the salt bridge network structure and thereby improve its thermal stability, it can be used to protect functional factors such as curcumin from thermal degradation during heat processing, and can be used in fields such as food, health products, and pharmaceuticals.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A method for preparing highly heat-resistant ferritin and its application, characterized by comprising the following steps:

[0007] a) Change the sequence at the triple axis position of ferritin to a sequence in which 1 - 3 acidic amino acids and basic amino acids appear alternately;

[0008] b) Introduce the ferritin mutant plasmid into competent cells;

[0009] c) Select the successfully mutated single colonies, culture, amplify, and induce the expression of mutant ferritin;

[0010] d) Lyse the cells with ultrasonic waves, and remove the miscellaneous proteins by centrifugation after heat treatment.

[0011] e) Pass the protein obtained in step d through a membrane, and prepare a mutant ferritin with a purity of more than 95% by column chromatography.

[0012] 2. A ferritin prepared by the method according to claim 1, characterized in that: the acidic amino acid is glutamic acid or aspartic acid, and the basic amino acid is arginine or lysine.

[0013] 3. A ferritin prepared by the method according to claim 1, characterized in that: the competent cells are generally common engineering bacteria such as Escherichia coli, yeast, Bacillus subtilis, etc.

[0014] 4. A ferritin prepared by the method according to claim 1, characterized in that: the heat treatment is carried out at 70 - 80 °C for 10 - 15 minutes, and the centrifugation treatment is carried out at 8000 - 10000 revolutions per minute for 5 - 10 minutes.

[0015] 5. A ferritin prepared by the method according to claim 1, characterized in that: the column chromatography is an anion column exchange column.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] In recent years, due to its unique structure and nanoscale size, ferritin has received great interest in the encapsulation, delivery, and sustained release of nutrients or drugs. Currently, a wide variety of bioactive small molecules, especially poorly water-soluble molecules such as β-carotene, curcumin, rutin, and photosensitizers, have been encapsulated in the cage of ferritin to improve their water solubility or stability. However, the heat resistance of natural ferritin is limited. How to further improve its heat resistance is of great significance for the application of functional factors such as curcumin in the food processing industry, and further broadens the application scope of ferritin nanocarriers in the food industry and medicine. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is the differential scanning calorimetry curve of the mutant obtained by mutating the human ferritin prepared in the present invention along the three-fold axis.

[0018] Example 1

[0019] 1. Synthesize the human ferritin H chain sequence, mutate it to EK at its three-fold axis position (positions 131 - 132), integrate the sequence with the Pet3a sequence, and introduce it into Escherichia coli competent cells;

[0020] 2. Pick positive single colonies, activate them in a medium, then amplify them in a large Erlenmeyer flask, and then add an inducer to induce the expression of the mutant protein;

[0021] 3. Centrifuge to obtain the bacterial cells, resuspend them in buffer and then disrupt by sonication. Heat the disrupted solution at 75 °C for 10 minutes, then centrifuge at 8000 r for ten minutes to remove the precipitate;

[0022] 4. After salting out and filtering the supernatant, pass it through an anion column to prepare the human ferritin mutant, named HuHF 3F -pos-neg 。

[0023] Example 2

[0024] Same as Example 1, but mutated to EKEKEK at positions 129 - 134.

[0025] Example 3

[0026] Same as Example 1, but use the soybean ferritin sequence.

[0027] Comparative Example 1

[0028] Same as Example 1, but mutated to A at the triple axis position.

[0029] Comparative Example 2

[0030] Same as Example 1, but without heat treatment during the extraction process.

[0031] Comparative Example 3

[0032] Same as Example 1, but use a cation exchange column during the extraction process.

[0033] Performance investigation

[0034] 1. The thermal denaturation temperature of the mutants in the examples and comparative examples was measured by differential scanning calorimetry (DSC). First, degas the protein sample at 0.5 mg / mL, then pipette 600 μL of the protein sample into the nano-DSC instrument. After equilibrating at 60 °C for 10 min, heat it at a heating scan rate of 1 °C / min until 115 °C. As Figure 1 shown, the thermal denaturation temperature of human ferritin in Example 1 was about 92 °C, which was about 6 °C higher than that of natural ferritin. The thermal denaturation temperature of the mutants in other examples also increased accordingly. In Comparative Example 1, since the salt bridge effect at the triple axis position was not increased, the thermal denaturation temperature did not change significantly.

[0035] 2. Heat treatment can improve the extraction efficiency of ferritin, especially for the mutants with improved thermal denaturation problems. Therefore, for the comparative example samples without heat treatment, due to more impurity proteins, the subsequent extraction effect will be affected.

[0036] The surface of the modified ferritin is mainly negatively charged and needs to be purified using an anion exchange column. A cation purification column cannot achieve the purification effect.

Claims

1. A method for preparing highly heat-resistant ferritin, characterized in that: The steps include: a) changing the sequence at the ferritin three-fold axis to a sequence in which 1-3 acidic and basic amino acids alternate; b) introducing the ferritin mutant plasmid into competent cells; c) selecting a single colony with successful mutation, culturing, amplifying, and inducing expression of mutant ferritin; d) The cells were disrupted by ultrasound, and then centrifuged to remove foreign proteins after heat treatment. e) The protein obtained in step d is passed through a membrane and subjected to column chromatography to obtain mutant ferritin with a purity of more than 95%.

2. A ferritin prepared according to the method of claim 1, characterized in that: The acidic amino acid is glutamic acid or aspartic acid, and the basic amino acid is arginine or lysine.

3. A ferritin prepared according to the method of claim 1, characterized in that: The competent cells are generally commonly used engineering bacteria such as Escherichia coli, yeast, and Bacillus subtilis.

4. A ferritin prepared according to the method of claim 1, characterized in that: The heating treatment is performed at 70-80° C. for 10-15 minutes, and the centrifugation treatment is performed at 8000-10000 rpm for 5-10 minutes.

5. A ferritin prepared according to the method of claim 1, characterized in that: The column chromatography is an anion exchange column.

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