High-specific-activity manganese superoxide dismutase as well as preparation method and application thereof

Abstract

The invention relates to high-specific-activity manganese superoxide dismutase as well as a preparation method and application thereof. The preparation method comprises the following steps: respectively preparing an AliSOD aqueous solution, a 2-methylimidazole aqueous solution and a zinc acetate aqueous solution; the method comprises the following steps: adding an AliSOD aqueous solution into a reaction container, sequentially adding a 2-methylimidazole aqueous solution and a zinc acetate aqueous solution under an ultrasonic condition, and then incubating for 20-30 minutes at room temperature and 180-220 r / min to obtain a mixed solution; s4, centrifuging the mixed solution obtained in the step S4, re-suspending the precipitate with water, then centrifuging, and drying the obtained precipitate to obtain sphere-like AliSOD ZIF-8; according to the manganese superoxide dismutase prepared by the method, the surfaces of zymoprotein are uniformly coated with a metal organic framework material, and the manganese superoxide dismutase and zymoprotein are applied as a feed additive, so that the activity can be kept in the stomach and intestine of an animal, and SOD is promoted to play a role through slow release, so that the intestinal health and production performance of the animal are improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to a high-specific-activity manganese superoxide dismutase and a preparation method and application thereof. Background Art

[0002] When livestock and poultry are affected by environmental, physiological and dietary factors, they will produce a large number of free radicals such as reactive oxygen species (ROS), which will lead to an imbalance between oxidation and antioxidant effects in the body, produce oxidative stress reactions, and thus affect the physiological functions and production performance of animals, causing huge economic losses and bringing serious harm to the stable development of animal husbandry. Feed enzyme preparations have been proven to be able to partially replace antibiotics. Oxidoreductases have become important candidate enzymes due to their own catalytic properties and multiple functions such as protecting animal health, promoting digestion and absorption, and improving the body's immunity. Among them, superoxide dismutase (SOD) is an important antioxidant enzyme in animals. It can effectively reduce animal oxidative stress, improve immunity, promote growth and prevent diseases. It can be used as a new enzyme preparation to replace antibiotics. In animal husbandry, adding SOD to livestock and poultry feed can prevent and alleviate oxidative stress caused by negative effects such as environmental stress, malnutrition, bacterial or viral invasion, remove free radicals in the body, enhance the body's antioxidant capacity and immunity, and improve the survival rate and health of young animals. According to the different metal cofactors bound to the active center of SOD, it is mainly divided into four types, namely CuZnSOD, FeSOD, MnSOD and NiSOD. Among them, MnSOD mainly exists in mitochondria, which are important places for cell oxidative metabolism and production of free radicals. It plays the most important regulatory role in anti-oxidative damage. At present, plant-derived and animal-derived MnSOD has been verified for related functions in cell and animal models, but it has defects such as low specific activity, high cost, poor thermal stability, and no resistance to pepsin, which leads to many bottlenecks in its industrial application as a feed additive in the feed field. Summary of the invention

[0003] Based on the above problems existing in the prior art, the present invention provides a method for preparing manganese superoxide dismutase with high specific activity. The manganese superoxide dismutase prepared by the method has a metal-organic framework material uniformly coated on the surface of the enzyme protein. When used as a feed additive, it can maintain activity in the gastrointestinal tract of animals and promote the function of SOD through sustained release, thereby improving the intestinal health and production performance of animals.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows:

[0005] A method for preparing high specific activity manganese superoxide dismutase comprises the following steps:

[0006] S1, dissolving AliSOD in water to obtain an AliSOD aqueous solution;

[0007] S2, dissolving 2-methylimidazole in water to obtain a 2-methylimidazole aqueous solution;

[0008] S3, dissolving zinc acetate in water to obtain a zinc acetate aqueous solution;

[0009] S4, adding the AliSOD aqueous solution into a reaction container, adding the 2-methylimidazole aqueous solution and the zinc acetate aqueous solution in sequence under ultrasonic conditions, and then incubating at room temperature and 180-220 r / min for 20-30 min to obtain a mixed solution;

[0010] S5. Centrifuge the mixed solution obtained in step S4, resuspend the precipitate in water and centrifuge again, and dry the precipitate to obtain spherical AliSOD@ZIF-8.

[0011] In some embodiments, in step S4, the volume ratio of the AliSOD aqueous solution, the 2-methylimidazole aqueous solution and the zinc acetate aqueous solution is 1:1:1.

[0012] In some embodiments, the concentration of the AliSOD aqueous solution is 0.5-2 mg / mL; the concentration of the 2-methylimidazole aqueous solution is 12-18 mg / mL; and the concentration of the zinc acetate aqueous solution is 8-12 mg / mL.

[0013] In some embodiments, step S5 is specifically as follows: after the mixed solution is centrifuged at 8000-10000 r / min for 10-15 min, the precipitate is resuspended and rinsed with water, and then centrifuged at 8000-10000 r / min for 10-15 min, and the obtained precipitate is dried.

[0014] In some embodiments, the AliSOD is a SOD gene obtained from an Alicyclobacillus sp. strain and obtained by exogenous expression, and the sequence of the SOD gene is shown in SEQ ID NO.1.

[0015] In some embodiments, the method for exogenously expressing the SOD gene comprises the following steps:

[0016] The SOD gene is introduced into an expression vector to construct a recombinant expression vector, which is then introduced into a host cell for culture to obtain AliSOD.

[0017] In some embodiments, the preparation of AliSOD comprises the following steps:

[0018] (1) The SOD gene of the Alicyclobacillus sp. strain was cloned and introduced into E. coli (BL21) to construct an expression system to obtain BL21 (DE3) / Pet30a (+) / SOD;

[0019] (2) BL21(DE3) / Pet30a(+) / SOD was transferred to LB medium containing kanamycin and placed in a constant temperature shaking incubator at 37°C and 220 rpm for 12-16 h for activation culture;

[0020] (3) The activated bacterial culture was transferred into LB medium containing kanamycin with a volume 100 times the volume of the culture medium and placed in a constant temperature shaking incubator at 37°C and 220 r / min to expand the culture until the OD 600 0.6-0.8;

[0021] (4) Add 0.6 mM IPTG and 1 mM MnSO to the container after expansion culture. 4 , placed in a constant temperature shaking incubator at 37°C, 220r / min for 6-8h;

[0022] (5) After induction, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and resuspend the cells in an appropriate amount of magnetic bead binding buffer;

[0023] (6) After resuspension, the cells were placed in an ice-water bath for ultrasonic cell disruption. The ultrasonic cycle conditions were: power ratio 40%, ultrasonication for 6 seconds, interval 10 seconds, and a total of 25 minutes;

[0024] (7) After the ultrasound, the cell lysate was centrifuged at 10,000 r / min for 10 min, and the supernatant was collected as the crude enzyme solution rich in AliSOD;

[0025] (8) The supernatant in step (7) was heated in a water bath at 70° C. for 5 min, and then centrifuged again at 10,000 rpm for 10 min. The supernatant was collected again to obtain a crude enzyme solution rich in AliSOD and less in impurities;

[0026] (9) Purify the crude enzyme solution rich in AliSOD and less in impurity proteins obtained in step (8) using His-tagged protein purification magnetic beads, and freeze-dry to obtain a solid AliSOD product.

[0027] The present invention also provides a manganese superoxide dismutase with high specific activity obtained by the preparation method of any of the above embodiments.

[0028] The present invention also provides the use of the high specific activity manganese superoxide dismutase as an animal feed additive.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention prepares spherical AliSOD@ZIF-8 with high specific activity and strong acid resistance by strictly controlling reaction process conditions, and uniformly wraps ZIF-8 on the surface of enzyme protein so that the obtained manganese superoxide dismutase has the characteristics of high stability and good gastric acid resistance. When used as a feed additive, it can ensure that AliSOD remains active in the gastrointestinal tract of animals, and promotes the function of AliSOD through sustained release, thereby effectively improving the intestinal health of animals.

[0031] In addition, the preparation method of the present invention is simple to operate, requires little equipment investment, and is suitable for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The electrophoresis diagram of the AliSOD protein prepared in Example 1;

[0033] Figure 2 is the SEM image of AliSOD@ZIF-8 prepared in Example 2;

[0034] Figure 3 The pH tolerance test results of AliSOD@ZIF-8;

[0035] Figure 4 The tolerance of AliSOD@ZIF-8 in simulated gastrointestinal fluid;

[0036] Figure 5 is the body weight of mice under different treatments;

[0037] Figure 6 This is the jejunal tissue section of the LSOD@ZIF-8 group. DETAILED DESCRIPTION

[0038] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] Example 1

[0041] A method for preparing a thermostable manganese superoxide dismutase comprises the following steps:

[0042] (1) Clone the SOD gene of Alicyclobacillus sp. strain (MCL6452249.1), whose sequence is shown in SEQ ID NO 1, and construct an E. coli (BL21) expression system;

[0043] (2) BL21(DE3) / Pet30a(+) / AliSOD was transferred to LB medium (containing 50 μg / mL kanamycin) and placed in a constant temperature shaking incubator at 37°C and 220 r / min for activation culture for 12-16 h;

[0044] (3) The activated bacterial culture was transferred into LB medium (containing 50 μg / mL kanamycin) with a volume 100 times that of the culture medium and placed in a constant temperature shaking incubator at 37°C and 220 r / min for 3 h until the OD 600 0.6-0.8;

[0045] (4) Add 0.6 mM IPTG and 1 mM MnSO to the container after expansion culture. 4 , placed in a constant temperature shaking incubator at 37°C, 220r / min for 6-8h;

[0046] (5) After induction, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and resuspend the cells in an appropriate amount of magnetic bead binding buffer;

[0047] (6) After resuspension, the cells were placed in an ice-water bath for ultrasonic cell disruption. The ultrasonic cycle conditions were: power ratio 40%, ultrasonication for 6 seconds, interval 10 seconds, and a total of 25 minutes;

[0048] (7) After the ultrasound, the cell lysate was centrifuged at 10,000 r / min for 10 min, and the supernatant was collected as the crude enzyme solution rich in AliSOD;

[0049] (8) The supernatant in step (7) was heated in a water bath at 70° C. for 5 min, and then centrifuged again at 10,000 rpm for 10 min. The supernatant was collected again to obtain a crude enzyme solution rich in AliSOD and less in impurities;

[0050] (9) The crude enzyme solution rich in AliSOD and less in impurities was purified using His-tagged protein purification magnetic beads, and freeze-dried to obtain a solid AliSOD product, the electrophoresis pattern of which is shown in FIG. Figure 1 shown.

[0051] Example 2

[0052] A method for preparing high specific activity manganese superoxide dismutase comprises the following steps:

[0053] S1. Dissolve the AliSOD prepared in Example 1 in pure water to prepare a 1 mg / mL AliSOD aqueous solution;

[0054] S2, dissolving 2-methylimidazole in purified water to prepare a 15.68 mg / mL 2-methylimidazole aqueous solution;

[0055] S3, dissolving zinc acetate in pure water to prepare a zinc acetate aqueous solution with a concentration of 10.48 mg / mL;

[0056] S4, measuring the AliSOD aqueous solution into a conical flask, and then placing it in an ultrasonic cleaning machine for ultrasonic treatment for 10 minutes, adding an equal volume of 2-methylimidazole aqueous solution to the AliSOD aqueous solution, and then adding an equal volume of zinc acetate aqueous solution to the AliSOD aqueous solution under ultrasonic conditions, and finally immediately placing it in a shaker at room temperature and 180-220 r / min for incubation for 20-30 minutes to obtain a mixed solution;

[0057] S5. Seal the mixed solution obtained in step S4, centrifuge at 10000 r / min for 12 min, resuspend and rinse the obtained precipitate with pure water, centrifuge again at 10000 r / min for 10 min, and dry the precipitate to obtain AliSOD@ZIF-8.

[0058] The obtained AliSOD@ZIF-8 was characterized by SEM, and the characterization results are as follows Figure 2 shown.

[0059] like Figure 2 AliSOD@ZIF-8 is a nearly spherical nanoparticle with a regular morphology and a particle size between 1000-3000nm.

[0060] The activity of AliSOD and AliSOD@ZIF-8 was determined by the pyrogallol method. The specific steps are as follows:

[0061] Prepare solution A: prepare 0.1 mol / L Tris-HCL buffer at pH 8.2, containing 0.001 mol / L EDTA solution;

[0062] Prepare Solution B: Add pyrogallol to a 0.01 mol / L hydrochloric acid solution to prepare a 0.0045 mol / L pyrogallol solution;

[0063] A blank group and an experimental group were set up in a 25°C water bath. For the blank group, 2.35 mL of solution A, 2 mL of distilled water, and 0.15 mL of solution B were added to a 10 mL glass test tube in sequence; for the experimental group, 2.35 mL of solution A, 1.98 mL of distilled water, 0.02 mL of enzyme solution, and 0.15 mL of solution B were added to a 10 mL colorimetric tube in sequence; after adding solution B, the mixture was immediately shaken and mixed, and the mixture was poured into a colorimetric dish. The initial and 1 minute absorbance values ​​of the blank group and the experimental group were measured under a wavelength of 325 nm, and the difference between the two was the self-oxidation rate of pyrogallol. The concentration of the enzyme solution was adjusted to make the self-oxidation rate of pyrogallol in the experimental group half of that in the blank group, and the sample activity (U / mL) was calculated.

[0064] The amount of SOD required to inhibit the auto-oxidation rate of pyrogallol by 50% at 25°C was defined as 1U.

[0065] It was calculated that the enzyme activity of AliSOD@ZIF-8 at 25°C and pH 7.2 was approximately 200 U / mg as determined by the pyrogallol self-oxidation method.

[0066] like Figure 3 The relative enzyme activities of AliSOD@ZIF-8 after treatment at pH 2.0, pH 3.0, and pH 4.0 for 1 hour were 33.9%, 115.6%, and 116.1%, respectively; while the relative enzyme activities of AliSOD without metal-organic framework mediation after treatment at pH 2.0, pH 3.0, and pH 4.0 for 1 hour were 0.0%, 5.1%, and 6.7%, respectively; compared with free AliSOD, AliSOD@ZIF-8 showed better enzyme catalytic activity in a strong acid environment, indicating that SOD@ZIF-8 constructed by the bionic mineralization strategy has better strong acid tolerance.

[0067] Example 3

[0068] The activity of SOD in simulated gastrointestinal fluid was detected. The specific implementation steps are as follows:

[0069] Prepare simulated gastric fluid: take 33.33 mg of pepsin (porcine source, 1:3000), 0.1 g of sodium chloride and 0.35 mL of 37% hydrochloric acid solution, add enough water to adjust the pH to 2.0, and make up to 50 mL.

[0070] Prepare simulated intestinal fluid: take 20 mg trypsin (porcine source, 1:250) and 0.68 g KH 2 PO 4 After adding enough water, adjust the pH to 7.0 with 0.4% NaOH and make up to 50 mL.

[0071] AliSOD sample and AliSOD@ZIF-8 sample with a volume ratio of 1:3 were taken and mixed with simulated gastric fluid, and the residual SOD activity was measured after 30min, 60min and 120min in a shaking water bath at 37℃. Then, the mixture treated with simulated gastric fluid for 120min was mixed with simulated intestinal fluid at a volume ratio of 1:2 and incubated in a water bath at 37℃ for 120min, and the residual SOD activity was finally measured.

[0072] like Figure 4 , the relative enzyme activity of AliSOD@ZIF-8 after treatment with simulated gastric and intestinal fluid was 90.7%, while that after treatment with AliSOD was 0.0%.

[0073] Example 4

[0074] In this example, a mouse feeding experiment was carried out using AliSOD@ZIF-8 as the material, and the specific steps are as follows:

[0075] (1) Feeding test groups: control group (Group A), low-dose SOD group (Group B), medium-dose SOD group (Group C), and high-dose SOD group (Group D). Each group had 10 replicates.

[0076] (2) Feeding experiment operation: The weight changes of mice were recorded every day from the beginning of the experiment (weighing at 8:30 am every day); the mice were gavaged at 9:00 am every other day (gavage every other day, a total of 10 times) and fed for 21 days.

[0077] Group A: Each mouse was gavaged with 200 μL of normal saline every day;

[0078] Group B: 200 μL AliSOD@ZIF-8 (15.5 mg / kg) was administered intragastrically every day;

[0079] Group C: 200 μL AliSOD@ZIF-8 (30.9 mg / kg) was administered intragastrically every day;

[0080] Group D: 200 μL AliSOD@ZIF-8 (61.9 mg / kg) was administered intragastrically every day;

[0081] On the 21st day, the mice were dissected to measure the length of the colon, and the jejunum was fixed (fixed in 10% paraformaldehyde) to detect the morphological structure of the intestinal villi.

[0082] The mouse body weight results were as follows Figure 5 shown. Figure 5 The results showed that the body weight of the low-dose AliSOD@ZIF-8 group had no significant difference with that of the control group, and did not affect the normal growth of mice; jejunal tissue sections ( Figure 6)The results showed that AliSOD@ZIF-8 significantly reduced the crypt depth of mice and significantly improved the jejunal morphology (V / C) of mice (as shown in Table 1).

[0083] Table 1 Morphological structure of jejunum in mice treated with different methods

[0084]

[0085] The above studies show that AliSOD@ZIF-8 can improve the intestinal health of animals, and the improvement of the jejunal morphology and structure of mice in the low-dose group (LSOD@ZIF-8) is the most obvious.

[0086] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a high specific activity manganese superoxide dismutase, characterized in that: The following steps are involved: S1, dissolving AliSOD in water to obtain an AliSOD aqueous solution; S2, dissolving 2-methylimidazole in water to obtain a 2-methylimidazole aqueous solution; S3, dissolving zinc acetate in water to obtain a zinc acetate aqueous solution; S4, adding the AliSOD aqueous solution into a reaction container, adding the 2-methylimidazole aqueous solution and the zinc acetate aqueous solution in sequence under ultrasonic conditions, and then incubating at room temperature and 180-220 r / min for 20-30 min to obtain a mixed solution; S5. Centrifuge the mixed solution obtained in step S4, resuspend the precipitate in water and centrifuge again, and dry the precipitate to obtain spherical AliSOD@ZIF-8.

2. The method for preparing high specific activity manganese superoxide dismutase according to claim 1, characterized in that: In step S4, the volume ratio of the AliSOD aqueous solution, the 2-methylimidazole aqueous solution and the zinc acetate aqueous solution is 1:1:

1.

3. The method for preparing high specific activity manganese superoxide dismutase according to claim 1, characterized in that: The concentration of the AliSOD aqueous solution is 0.5-2 mg / mL; the concentration of the 2-methylimidazole aqueous solution is 12-18 mg / mL; and the concentration of the zinc acetate aqueous solution is 8-12 mg / mL.

4. The method for preparing high specific activity manganese superoxide dismutase according to claim 1, characterized in that: Step S5 is specifically as follows: after the mixed solution is centrifuged at 8000-10000 r / min for 10-15 min, the precipitate is resuspended and washed with water, and then centrifuged at 8000-10000 r / min for 10-15 min, and the obtained precipitate is dried.

5. The method for preparing high specific activity manganese superoxide dismutase according to any one of claims 1 to 4, characterized in that: The AliSOD is obtained by exogenously expressing the SOD gene obtained from the Alicyclobacillus sp. strain, and the sequence of the SOD gene is shown in SEQ ID NO.

1.

6. The method for preparing high specific activity manganese superoxide dismutase according to claim 5, characterized in that: The method for exogenously expressing the SOD gene comprises the following steps: The SOD gene is introduced into an expression vector to construct a recombinant expression vector, which is then introduced into a host cell for culture to obtain AliSOD.

7. The method for preparing high specific activity manganese superoxide dismutase according to claim 6, characterized in that: The specific steps include: (1) The SOD gene of the Alicyclobacillus sp. strain was cloned and introduced into E. coli (BL21) to construct an expression system to obtain BL21 (DE3) / Pet30a (+) / SOD; (2) BL21(DE3) / Pet30a(+) / SOD was transferred to LB medium containing kanamycin and placed in a constant temperature shaking incubator at 37°C and 220 rpm for 12-16 h for activation culture; (3) After activation, the bacterial suspension was transferred into LB medium containing kanamycin with a volume 100 times the volume of the culture medium and placed in a constant temperature shaking incubator at 37°C and 220 r / min to expand the culture until the OD 600 0.6-0.8; (4) Add 0.6 mM IPTG and 1 mM MnSO4 to the container after expansion culture, and place it in a constant temperature shaking incubator at 37°C and 220 r / min for 6-8 h; (5) After induction, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and resuspend the cells in an appropriate amount of magnetic bead binding buffer; (6) After resuspension, the cells were placed in an ice-water bath for ultrasonic cell disruption. The ultrasonic cycle conditions were: power ratio 40%, ultrasonication for 6 seconds, interval 10 seconds, and a total of 25 minutes; (7) After the ultrasound, the cell lysate was centrifuged at 10,000 r / min for 10 min, and the supernatant was collected as the crude enzyme solution rich in AliSOD; (8) The supernatant in step (7) was heated in a water bath at 70° C. for 5 min, and then centrifuged again at 10,000 rpm for 10 min. The supernatant was collected again to obtain a crude enzyme solution rich in AliSOD and less in impurities; (9) Purify the crude enzyme solution rich in AliSOD and less in impurity proteins obtained in step (8) using His-tagged protein purification magnetic beads, and freeze-dry to obtain a solid AliSOD product.

8. The high specific activity manganese superoxide dismutase obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the high specific activity manganese superoxidase according to claim 8 as an animal feed additive.

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