Preparation method of metal ion induced phycocyanin shell-core structure

Abstract

The invention discloses a preparation method of a metal ion induced phycocyanin shell-core structure, which comprises the following steps: S1, dissolving cyanobacteria protein in water to prepare a cyanobacteria protein solution; s2, CuCl2. 2H2O is dissolved in water, and a metal ion mother solution is prepared; and S3, mixing the cyanobacteria protein solution prepared in the step S1 and the metal ion mother solution prepared in the step S2 in a dark place at 10-30 DEG C to prepare the metal ion induced phycocyanin shell-core structure. The core-shell structure has a typical alpha-spiral structure, the diameter of the spherical structure is 10-12 nm in a TEM result, the problem that phycocyanin is prone to denaturation is solved, and the purpose of enhancing the illumination stability and the heat stability of the phycocyanin is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of protein modification, in particular to a method for preparing a metal ion-induced phycocyanin shell-core structure. Background Art

[0002] Phycocyanin is one of the rare pigment proteins in nature. It is not only brightly colored, but also a nutritious protein in itself. It has a complete amino acid composition and a high content of essential amino acids. It has high color and purity, and does not produce bad odor or taste. Phycocyanin is another type of photosynthetic pigment protein besides chlorophyll a. It is composed of phycobilin (open-chain linear tetrapyrrole compound) as a cofactor and a Cys-containing apoprotein bound by a thioether bond. Phycobilin molecules interact with aromatic amino acids on the surrounding connecting proteins, changing the energy state of the chromogenic molecules and affecting the structure and function of phycocyanin. Studies have shown that phycocyanin (blue, absorbs orange light) can form cyclic polymers or (α 3 β 3 ), the α- and β-subunits form an αβ heterodimer, which then assembles to form (αβ) 6 or (α 3 β 3 ) hexamer, which is the basic unit of the hierarchical assembly of phycocyanin.

[0003] The research of domestic and foreign scholars mainly focuses on the structure of natural phycocyanin. Its chromophore is an open-chain structure composed of four substituted pyrrole rings connected by carbon atoms, and is connected to the apoprotein through a thioether bond. Changes in the system environment will change the folding mode of natural phycocyanin. The chromophore is the main functional group that causes phycocyanin to be brightly colored. The unshared electron pair on the pyrrole N forms a conjugated system with the apoprotein side chain. Under the influence of the external environment, the change in the spatial structure of the apoprotein leads to a change in the electron cloud density around the open-chain tetrapyrrole structure chromophore, and the ultraviolet absorption and characteristic fluorescence of phycocyanin change. Protein is a high molecular compound. In a non-isoelectric solution system, its surface carries a charge and will hydrate with water to form a hydration film. Studies have shown that enhancing the rigidity of proteins, reducing the entropy change in the protein folding process, reducing the tension of protein conformation, increasing the α-helical dipole, improving the protein encapsulation rate, and reducing the hydrophobic surface area accessible to the solvent can all improve the stability of proteins.

[0004] Phycocyanin has important economic potential in the pharmaceutical, biomedical and food industries. However, sensitivity to temperature has become a key issue that hinders the widespread industrial application of phycocyanin. Temperatures above 45°C at neutral pH will cause phycocyanin to denature, resulting in the disappearance of its blue color. Therefore, it is necessary to improve the stability of phycocyanin. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a method for preparing a metal ion-induced phycocyanin shell-core structure. The present invention regulates the microscopic morphology of phycocyanin after metal ion induction, so that shell-core structures with different morphologies can be prepared under the conditions of specific temperature and specific ionic strength.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a method for preparing a metal ion-induced phycocyanin shell-core structure, comprising the following steps:

[0008] S1, dissolving cyanobacterial protein in water to prepare a cyanobacterial protein solution;

[0009] S2, CuCl 2 ·2H 2 O is dissolved in water to obtain a metal ion mother solution;

[0010] S3, in a dark environment at 10-30° C., mixing the cyanobacterial protein solution prepared in S1 and the metal ion mother solution prepared in S2 to prepare the metal ion-induced cyanobacterial protein shell-core structure.

[0011] In one embodiment of the present invention, the measured purity of the cyanobacterial protein is: A620 nm / A280 nm>2.0.

[0012] In one embodiment of the present invention, the cyanobacterial protein is a commercially available product of Zhejiang Binmei Co., Ltd.

[0013] In one embodiment of the present invention, in S1, the concentration of the cyanobacterial protein solution is 10-30 μM, and the pH of the system is adjusted to 7.0.

[0014] In one embodiment of the present invention, in S2, the metal ion mother solution CuCl 2 The concentration is 10-50mM.

[0015] In one embodiment of the present invention, in S3, the cyanobacterial protein solution and the metal ion mother solution are mixed at a volume ratio of 9:1, and finally, the molar ratio of ions to proteins is 50-250:1.

[0016] In one embodiment of the present invention, in S3, the mixing is performed under light-proof conditions.

[0017] The second object of the present invention is to provide a metal ion-induced phycocyanin core-shell structure obtained by the above preparation method.

[0018] In one embodiment of the present invention, the product is used directly without post-treatment.

[0019] In one embodiment of the present invention, the metal ion-induced phycocyanin shell-core structure is a three-dimensional spherical particle with an average diameter of 10-12 nm.

[0020] The third object of the present invention is to provide an application of the metal ion-induced phycocyanin shell-core structure as a protein stabilizer.

[0021] The beneficial technical effects of the present invention are:

[0022] The present invention is the first to regulate the microscopic morphology of phycocyanin after metal ion induction, so that shell-core structures with different morphologies can be prepared under the conditions of specific temperature and specific ion strength.

[0023] The core-shell structure obtained by the present invention broadens the idea of ​​improving the stability of phycocyanin in the field of protein processing and modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a negatively stained transmission electron microscope (TEM) image of Example 1;

[0025] Figure 2 It is the negative staining transmission electron microscope (TEM) image of Example 2;

[0026] Figure 3 It is the negative staining transmission electron microscope (TEM) image of Example 3;

[0027] Figure 4 It is the negative staining transmission electron microscope (TEM) image of comparative example 1;

[0028] Figure 5 It is the negative staining transmission electron microscope (TEM) image of comparative example 2;

[0029] Figure 6 It is the negative staining transmission electron microscope (TEM) image of comparative example 3;

[0030] Figure 7 Circular dichroism spectra (CD) of Example 1, Example 2, Example 3 and Comparative Example 4;

[0031] Figure 8 is the secondary structure content of Example 1, Example 2, Example 3 and Comparative Example 4;

[0032] Fig. 9 The infrared spectra (FT-IR) of Example 1, Example 2, Example 3 and Comparative Example 4 are shown;

[0033] Fig.10 The ultraviolet spectra (UV) of Example 1, Example 2, Example 3 and Comparative Example 4 are shown;

[0034] Fig.11 The fluorescence spectra (FL) of Example 1, Example 2, Example 3 and Comparative Example 4 are shown in FIG.

[0035] Fig.12 Schematic diagram of the formation of the core-shell structure in Example 2. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0037] The cyanobacterial protein in the examples of the present invention is a commercially available product of Zhejiang Binmei Co., Ltd., with a molar mass of 230 kDa; the measured purity is: A620 nm / A280 nm>2.0.

[0038] Embodiment 1:

[0039] A method for preparing a metal ion-induced phycocyanin shell-core structure comprises the following steps:

[0040] S1: dissolving phycocyanin in water at pH = 7.0 to obtain a protein solution with a concentration of 25 μM;

[0041] S2: CuCl 2 ·2H 2 O is dissolved in water to obtain a copper ion mother solution with a concentration of 50.0 mM;

[0042] S3: The protein solution obtained in S1 and the copper ion mother solution obtained in S2 were mixed at a volume ratio of 9:1 for 12 h at 20°C in the dark. The molar ratio of copper ion to protein was 250:1 (the concentration of phycocyanin was 20 μM, and the concentration of CuCl 2 ·2H 2 O concentration is 5 mM).

[0043] Embodiment 2:

[0044] A method for preparing a metal ion-induced phycocyanin shell-core structure comprises the following steps:

[0045] S1: dissolving phycocyanin in water at pH = 7.0 to obtain a protein solution with a concentration of 25 μM;

[0046] S2: CuCl 2 ·2H 2 O is dissolved in water to obtain a copper ion mother solution with a concentration of 30.0 mM;

[0047] S3: The protein solution obtained in S1 and the copper ion mother solution obtained in S2 were mixed at a volume ratio of 9:1 for 12 h at 20°C in the dark. The molar ratio of copper ion to protein was 150:1 (the concentration of phycocyanin was 20 μM, and the concentration of CuCl 2 ·2H2 O concentration was 3 mM).

[0048] Embodiment 3:

[0049] A method for preparing a metal ion-induced phycocyanin shell-core structure comprises the following steps:

[0050] S1: dissolving phycocyanin in water at pH = 7.0 to obtain a protein solution with a concentration of 25 μM;

[0051] S2: CuCl 2 ·2H 2 O is dissolved in water to obtain a copper ion mother solution with a concentration of 10.0 mM;

[0052] S3: The protein solution obtained in S1 and the copper ion mother solution obtained in S2 were mixed at a volume ratio of 9:1 for 12 h at 20°C in the dark, and the molar ratio of copper ion to protein was 50:1 (the concentration of phycocyanin was 20 μM, and the concentration of CuCl 2 ·2H 2 O concentration is 1 mM).

[0053] Comparative Example 1:

[0054] A method for preparing a metal ion-induced phycocyanin complex comprises the following steps:

[0055] S1: dissolving phycocyanin in water at pH = 7.0 to obtain a protein solution with a concentration of 25 μM;

[0056] S2: ZnCl 2 Dissolve in water to obtain a zinc ion mother solution with a concentration of 50.0 mM;

[0057] S3: The protein solution obtained in S1 and the zinc ion mother solution obtained in S2 were mixed at a volume ratio of 9:1 for 12 h at 20°C in the dark, and the molar ratio of zinc ion to protein was 250:1 (the concentration of phycocyanin was 20 μM, and the concentration of ZnCl 2 The concentration is 5 mM).

[0058] Comparative Example 2:

[0059] A method for preparing a metal ion-induced phycocyanin complex comprises the following steps:

[0060] S1: dissolving phycocyanin in water at pH = 7.0 to obtain a protein solution with a concentration of 25 μM;

[0061] S2: CaCl 2 Dissolve in water to obtain a calcium ion stock solution with a concentration of 50.0 mM;

[0062] S3: The protein solution obtained in S1 and the calcium ion mother solution obtained in S2 were mixed at a volume ratio of 9:1 for 12 h at 20°C in the dark. The molar ratio of calcium ion to protein was 250:1 (the concentration of phycocyanin was 20 μM, and the CaCl 2 The concentration is 5 mM).

[0063] Comparative Example 3:

[0064] A method for preparing a phycocyanin solution comprises the following steps:

[0065] S1: dissolving phycocyanin in water at pH = 7.0 to obtain a protein solution with a concentration of 25 μM;

[0066] S2: At 20°C in the dark, dilute the protein solution obtained in S1 to 20 μM and let it stand for 12 h (equivalent to a molar ratio of ions to proteins of 0:1).

[0067] Detection test

[0068] (I) Take samples of Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3, and use a transmission electron microscope to analyze the appearance morphology: take 10 μL of the sample to be tested and drop it on a copper mesh with an ultra-thin carbon film, and then use filter paper to dry it after 5 minutes; drop 10 μL of uranyl acetate (2%) on the copper mesh and keep it for 5 minutes and then use filter paper to dry it; place it on a transmission electron microscope and use a voltage of 80 kV to observe the sample morphology. Observe the same sample in different areas to avoid experimental errors. The results are as follows: Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 shown. Figure 1 Under the transmission electron microscope of (Example 1), spherical structures of uniform size can be seen, and the spherical particles present a three-dimensional core-shell structure. After Image J analysis, the diameter of the spherical structure is about 11-12 nm. Figure 2 Under the transmission electron microscope of (Example 2), spherical structures of relatively uniform size can be seen, with a diameter of about 10-11 nm. Figure 3 The transmission electron microscope (TEM) shows a spherical particle structure, and the particles are smaller. Figure 4 (Comparative Example 1), Figure 5 (Comparative Example 2), Figure 6 The transmission electron microscopy image of (Comparative Example 3) shows that the spherical structure disappears and there are irregular aggregates or particles in the field of view.

[0069] (ii) Take samples of Example 1, Example 2, Example 3 and Comparative Example 3, and use J-1500 circular dichroism spectrometer to measure the secondary structure of samples of Example 1, Example 2, Example 3 and Comparative Example 3. After diluting the protein concentration to 0.2% (w / v), place it in a quartz cuvette with a light path of 1 mm, set the wavelength range to 190-260 nm at room temperature, and scan at a speed of 200 nm / min for 3 consecutive scans. Deionized water was used as a blank control, and Jasco-Corp software was used to calculate the secondary structure. The results are as follows: Figure 7 , Figure 8 As shown, before adding copper ions, the phycocyanin of comparative example 3 contained 55.9±3.19% of α-helical structure and 24.23±2.49% of β-folded structure. After the introduction of copper ions, the α-helical content in Example 3 was reduced to 54.67±3.88%, the β-folded structure remained basically unchanged (26.67±4.16%), and 0.533±0.41% of the β-turn structure was converted from part of the irregular curling. As the copper ion concentration continued to increase, the α-helical structure in Example 2 increased to 60.73±1.25%, the β-folded structure remained basically unchanged (26.67±4.16%), and the β-turn structure increased to 11.433±3.01%. The β-folded structure in Example 1 basically disappeared and was converted to 56.90±5.85% of the α-helical structure and 14.30±4.60% of the β-turn structure.

[0070] (III) Samples of Example 1, Example 2, Example 3 and Comparative Example 3 were taken and analyzed using PerkinElmer Fourier transform infrared spectroscopy (FTIR, Thermo Scientific Nicolet iS5) at 4000 cm -1 -400cm -1 The infrared spectrum at Fig. 9 As shown, the peptide bonds of Example 1, Example 2, Example 3 and Comparative Example 3 are at 1655 cm -1 and 1542cm -1 Characteristic absorption peak appears at 3400-3300cm -1 The absorption peaks of the bands indicate that -OH and -NH have appeared in the structure. Among them, the amide A band (3500-3200cm -1 ) is the main peak vibration range of NH stretching vibration. The appearance of this characteristic peak indicates the existence of hydrogen bonds or van der Waals interactions in the system.

[0071] (iv) Take samples of Example 1, Example 2, Example 3 and Comparative Example 3, dilute them into solutions with a protein concentration of 1 mg / mL, and perform scanning tests in the range of 190-750 nm using an ultraviolet-visible spectrometer. Fig.10 The ultraviolet spectra (UV) of Example 1, Example 2, Example 3 and Comparative Example 3 show that an absorption peak appears at 617 nm in Comparative Example 3. With the introduction of copper ions, the maximum absorption peak at this location in Example 3, Example 2 and Example 1 decreases and gradually disappears.

[0072] (V) Take samples of Example 1, Example 2, Example 3 and Comparative Example 3, dilute them into solutions with a protein concentration of 1 mg / mL, and characterize the fluorescence properties of the proteins and the microenvironmental changes of the side chain groups using a fluorescence spectrometer (Hitachi F-2700). Parameter settings: excitation wavelength is 620 nm, slit width is 5 nm, and the scanning range of the emission spectrum is 630-720 nm. Fig.11 The fluorescence spectra (FL) of Example 1, Example 2, Example 3 and Comparative Example 3 are shown. Comparative Example 3 has typical maximum absorption peaks at 562nm, 592nm, 615nm and 652nm, and the positive absorption in the range of 250-280nm is related to the absorption of aromatic amino acids such as tyrosine, tryptophan and phenylalanine. After the addition of copper ions, the ultraviolet absorption peak at 615-652nm moves from 647.5nm (Comparative Example 3) to 640.5nm (Example 2), blue-shifting, and the absorption intensity decreases.

[0073] The acidity of metal ions directly affects the formation of metal hydroxides / oxides. Metal ions with strong acidity have higher storage modulus and stronger hydrolysis ability. Fig.12 This is a schematic diagram of the formation of a core-shell structure of phycocyanin under the induction of a certain concentration of copper ions. In summary, it can be explained that after copper ions are introduced into the phycocyanin system, phycocyanin is induced to assemble, and the metal ions combine with pyrrole, amino acid side chain amino groups, carboxyl groups and other functional groups that are prone to coordination, thereby stabilizing the protein conformation and enhancing its light stability and thermal stability.

[0074] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a metal ion-induced phycocyanin core-shell structure, characterized in that: The preparation method comprises the following steps: S1, dissolving cyanobacterial protein in water to prepare a cyanobacterial protein solution; S2, dissolving CuCl2·2H2O in water to prepare a metal ion mother solution; S3, mixing the cyanobacterial protein solution prepared in S1 and the metal ion mother solution prepared in S2 at 10-30° C. to prepare the metal ion-induced cyanobacterial protein shell-core structure.

2. The preparation method according to claim 1, characterized in that: The measured purity of cyanobacterial protein was: A620 nm / A280nm>2.

0.

3. The preparation method according to claim 1, characterized in that: In S1, the concentration of the cyanobacterial protein solution is 10-30 μM, and the pH of the system is adjusted to 7.

0.

4. The preparation method according to claim 1, characterized in that: In S2, the concentration of CuCl2 in the metal ion mother solution is 10-50mM.

5. The preparation method according to claim 1, characterized in that: In S3, the cyanobacteria protein solution and the metal ion mother solution are mixed at a volume ratio of 9:1, and finally, the molar ratio of ions to proteins is 50-250:

1.

6. The preparation method according to claim 1, characterized in that: In S3, the mixing is performed under light-protected conditions.

7. A metal ion-induced phycocyanin core-shell structure obtained by the preparation method according to any one of claims 1 to 6.

8. The metal ion-induced phycocyanin core-shell structure according to claim 7, characterized in that: No post-processing required, just use directly.

9. The metal ion-induced phycocyanin core-shell structure according to claim 7, characterized in that: It is three-dimensional spherical particles with an average diameter of 10-12nm.

10. An application of the metal ion-induced phycocyanin core-shell structure according to claim 7, characterized in that: Used as a protein stabilizer.

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