Nano enzyme with SOD (superoxide dismutase) enzyme activity as well as preparation method and application thereof

Abstract

The invention relates to a nano-enzyme with SOD (superoxide dismutase) enzyme activity as well as a preparation method and application thereof. Specifically, the nano-enzyme with similar SOD (superoxide dismutase) enzyme activity is synthesized through an in-vitro synthesis or biomimetic synthesis method by utilizing the structural characteristics of ferritin. By utilizing the method disclosed by the invention, the nanometer enzyme with similar SOD enzyme activity can be mildly, conveniently, quickly and massively synthesized in a biosynthesis or in-vitro recombination manner.

Description

Technical Field

[0001] The present invention belongs to the cross-field of nanobiology, biomimetic synthesis and enzymology. In particular, the present invention relates to a nanozyme having SOD enzyme activity, a preparation method and its use. The present invention obtains a nanozyme having SOD-like enzyme activity by adding ferrous salts during the expression of ferritin from different species or by recombining ferrous salts and phosphates after purification through an in vitro synthesis or biomimetic synthesis method. Background Art

[0002] As a new material, nanozymes refer to inorganic nanomaterials with enzyme-like activity (reference 1). Nanozymes not only have the physical and chemical properties of nanomaterials themselves, but also have catalytic activity and reaction kinetics similar to natural enzymes. So far, researchers have synthesized a variety of nanozymes and are able to simulate different types of natural enzymes, including a variety of oxidoreductases, hydrolases, etc. (references 2-5). Compared with natural enzymes, nanozymes have the characteristics of high stability, easy modification and adjustable activity, making nanozymes, as a new type of simulated enzyme, play a very important role in environmental governance (reference 6), disease detection and treatment (reference 7) and other fields. With the deepening of research, the types of nanozymes have expanded from the initial magnetic particles to more than a thousand different types of nanozymes. At the same time, the emergence of nanozymes has also prompted researchers to explore the enzyme-like activity of natural biominerals. Natural biominerals are inorganic minerals synthesized by living organisms with special advanced structures and assembly methods, such as bones and teeth of animals. They are widely present in different living organisms, including bacteria, fungi, plants and animals. At present, three types of biominerals have been found to have peroxidase-like activity: magnetosomes in magnetic bacteria, ferrihydrite mineralized on the surface of fungi, and horse spleen ferritin (references 8-10), but these studies are still limited to specific species. As a widely existing biomineral, the activity relationship and catalytic mechanism of nanozymes in different species are still lacking. Ferritin, as a natural iron storage protein, is widely distributed in almost all species (reference 11). Based on their structural differences, ferritins in different species can be divided into DNA binding protein from starved cells (DNA binding protein from starved cells, Dps) in bacteria and archaea, heme-containing bacterioferritin (bacterial ferritin, Bfr) in bacteria, and conventional ferritin (FTn) widely present in bacteria, archaea and fungi (reference 11). Although the sequence compositions of these ferritins from different species are quite different, they can all store iron ions in their inner cavity in the form of inorganic iron cores, thus forming a natural inorganic nanomaterial, which makes ferritin an ideal material for studying the enzyme activity of nanobiominerals. At the same time, based on the stable core-shell structure of ferritin itself and its easy genetic engineering characteristics, many studies have used ferritin as a template to synthesize different types of ferritin nanozymes for tumor treatment, cell protection and other applications, indicating the huge application potential of ferritin nanozymes (reference 12). Therefore, the study of natural ferritin nanozymes can not only provide a new explanation for the concept and function of nanozymes, but also provide a theoretical basis for the synthesis and application of ferritin nanozymes.

[0003] Superoxide dismutase (SOD), also known as superoxide dismutase, is a class of enzymes that catalyze the production of superoxide anion free radicals (O2 - ) into H2O2 and O2. It is widely distributed and has been isolated from various organisms such as bacteria, fungi, algae, plants, protozoa, insects, fish and mammals. Superoxide dismutase has free radical scavenging, antioxidant and anti-aging effects, and is very useful in the treatment of diseases caused by superoxide free radicals (O2 - It has unique therapeutic effects on diseases caused by oxygen poisoning, acute inflammation, various autoimmune diseases, radiation sickness and senile cataracts, and is a promising new type of medicinal enzyme. Among them, copper-zinc SOD (Cu / Zn-SOD) is blue-green and mainly exists in the cytoplasm of eukaryotic cells.

[0004] The activity levels of ferritin-based superoxide dismutase (SOD) nanozymes from different biological sources are still unclear in the art, and there is still a need to obtain ferritin nanozymes with high levels of SOD enzyme activity through in vitro synthesis or biosynthesis or in vitro recombination methods. Summary of the invention

[0005] One aspect of the present invention relates to an isolated nanozyme having SOD activity, which is ferrihydrite containing phosphorus.

[0006] In some embodiments, the isolated nanozyme with SOD activity is isolated from the ferritin of archaea or bacteria, obtained by utilizing the ferrihydrite synthesis process, or synthesized in situ by adding ferrous salt during the expression of ferritin using the oxidase center and nucleation site of ferritin itself, or recombinantly synthesized in vitro by adding ferrous salt and phosphate to the solution system after the ferritin is purified, and it is a nanozyme with SOD activity contained in the ferritin shell or a nanozyme with SOD activity that does not contain a ferritin shell, wherein the isolated nanozyme with SOD activity is obtained by incorporating phosphate during the ferrihydrite synthesis process or by adding phosphate during the ferritin expression process or in the solution system.

[0007] In a further embodiment, the iron salt is a ferrous salt, more preferably, the metal salt is a ferrous sulfate salt.

[0008] In a further embodiment, the phosphate is potassium, sodium or ammonium phosphate, more preferably, the phosphate is potassium phosphate.

[0009] In a further embodiment, the ferritin is bacterial ferritin Bfr or conventional ferritin FTn or a mutant thereof, preferably, the ferritin is ferritin derived from archaea or bacteria, more preferably, the ferritin is ferritin derived from archaea, more preferably, the ferritin is ferritin derived from Sulfolobales or Pyrococcus, more preferably, the ferritin is encoded by a sequence selected from any one of SEQ ID NOs: 3-7 and 9-11, more preferably, the ferritin is encoded by a sequence selected from any one of SEQ ID NOs: 3-6.

[0010] In some embodiments, the isolated nanozyme with SOD activity is a nanozyme with SOD activity that does not contain a ferritin shell.

[0011] In some embodiments, the isolated nanozyme with SOD activity has a molar ratio of phosphorus to iron of 0.05-2.5, preferably, a molar ratio of phosphorus to iron of 0.1-2.0, more preferably, a molar ratio of phosphorus to iron of 0.2-1.5, more preferably, a molar ratio of phosphorus to iron of 0.3-1.2, more preferably, a molar ratio of phosphorus to iron of 0.5-1.0.

[0012] In some embodiments, the DLS particle size range of the isolated nanozyme with SOD activity is 0.1-100 nm, preferably, 0.2-50 nm, more preferably, 0.3-25 nm, more preferably, 0.4-20 nm, more preferably, 0.5-15 nm, more preferably, 0.6-10 nm, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 nm.

[0013] In another aspect, the present invention relates to a method for preparing the isolated nanozyme having SOD activity as described above, which is a method comprising the following steps:

[0014] Method 1: 1) Optionally, culturing archaea or bacteria and collecting bacterial cells; 2) disrupting archaea or bacteria in a buffer of pH 7-9.0, and centrifuging to remove insoluble matter and cell debris to obtain a supernatant; 3) for bacterial Bfr purification, incubating the supernatant at 60-68°C for 5-20 minutes, cooling and centrifuging at about 10000-18000 x g for about 5-50 minutes to remove insoluble matter, purifying the obtained supernatant with an anion purification column and molecular sieve, and collecting the bacterial Bfr fraction; for archaeal FTn purification, incubating the supernatant at 70-85°C for 5-30 minutes, cooling and purifying the supernatant with anion purification column and molecular sieve, and collecting the FTn fraction; and 4) optionally, removing the ferritin shell to obtain a nanozyme with SOD activity without the ferritin shell;

[0015] Method 2: a) quickly adding an aqueous solution of iron salt with a concentration of 0.01-0.1 M to an aqueous solution of phosphate with a concentration in the range of 0.001-0.2 M under rapid mixing; b) slowly adding a NaOH or KOH solution to adjust the pH to about 6.2-7.8 and reacting for 10 min-2 hr; and c) optionally, washing the precipitate with water by centrifugation and freeze-drying for later use;

[0016] Method 3: i) obtaining a buffer solution of bacterial ferritin Bfr or ferritin FTn with a concentration of 0.1-10 mg / mL; ii) adding a phosphate aqueous solution with a concentration of 2-80 mM to the solution of step i) and mixing; iii) slowly adding a ferrous salt aqueous solution with a concentration of 2-80 mM to the solution of step ii); and iv) optionally, centrifuging to remove the precipitate and desalting the supernatant; or

[0017] Method 4: I) optionally, obtaining recombinant cells heterologously expressing bacterial ferritin Bfr or ferritin FTn; II) culturing the recombinant cells heterologously expressing bacterial ferritin Bfr or ferritin FTn in a liquid culture medium to the logarithmic phase; III) adding a ferrous salt aqueous solution with a concentration of 0.5-10mM under the condition of inducing the expression of the bacterial ferritin Bfr or ferritin FTn and continuing to culture for 1-24hr; IV) purifying the bacterial ferritin Bfr or ferritin FTn from the induced expression recombinant cells; and V) optionally, removing the protein shell of the bacterial ferritin Bfr or ferritin FTn and obtaining a nanozyme that does not contain a protein shell.

[0018] A further aspect of the present invention relates to a composition comprising the isolated nanozyme having SOD activity as described above.

[0019] In some embodiments, the composition is a pharmaceutical composition, a cosmetic, or a toothpaste.

[0020] In another aspect, the present invention relates to use of the isolated nanozyme having SOD activity as described above in the preparation of an agent for scavenging superoxide radicals.

[0021] In another aspect, the present invention relates to the isolated nanozyme having SOD activity as described above, which is used for scavenging superoxide radicals.

[0022] In another aspect, the present invention relates to a method for scavenging superoxide radicals, comprising administering the isolated nanozyme having SOD activity as described above to a subject in need of scavenging superoxide radicals.

[0023] In another aspect, the present invention relates to the use of the isolated nanozyme having SOD activity as described above in the preparation of a medicament for preventing or treating conditions and / or diseases caused by superoxide radicals in a subject.

[0024] In another aspect, the present invention relates to the isolated nanozyme having SOD activity as described above, for use in preventing or treating conditions and / or diseases caused by superoxide radicals in a subject.

[0025] In another aspect, the present invention relates to a method for preventing or treating conditions and / or diseases caused by superoxide radicals in a subject, comprising administering a preventively or therapeutically effective amount of the isolated nanozyme having SOD activity as described above to a subject in need thereof.

[0026] In another aspect, the present invention relates to the use of the isolated nanozyme with SOD activity as a natural antioxidant.

[0027] In another aspect, the present invention relates to the use of the isolated nanozyme with SOD activity in enhancing the stress resistance of microorganisms or plants.

[0028] In other words, the present invention relates to the synthesis of ferritin superoxide dismutase (SOD) nanozymes from different biological sources, the verification of the active source and the identification of its related antioxidant physiological functions. Specifically, the present invention first adds ferrous salts during the expression of ferritin from different species by a biosynthetic method, and synthesizes SOD nanozymes in situ using the iron oxidase center and nucleation site of ferritin itself. Through activity comparison, elemental composition analysis, in vitro recombination verification, mutant construction and other means, the SOD enzyme activity of ferritin from different biological sources is identified to be different. The present invention finds that ferritin from different species sources will form iron cores with different iron-phosphorus contents due to their structural differences, thereby affecting its SOD enzyme activity, and finds that SOD enzyme activity is significantly correlated with species source, wherein ferritin from bacteria and archaea has higher SOD enzyme activity than human heavy chain ferritin nanozymes, and the activity difference is proved to be derived from the difference in the ratio of iron-phosphorus in the inner core by elemental content determination and in vitro recombination experiments; the difference in the inner core composition of ferritin nanozymes is further explained by the construction of ferritin mutants, which is determined by the protein structure. The present invention illustrates the effect of phosphate on the structure and activity of the iron core by the synthesis and characterization of 2-line ferrihydrite in the ferritin-like core. The present invention utilizes the above-mentioned ferritin nanozyme with high SOD enzyme activity to verify its ability to alleviate oxidative damage caused by superoxide anions in an Escherichia coli transgenic model. The present invention also synthesizes ferritin nanozymes with high SOD enzyme activity in vitro in combination with the structural and activity characteristics of the above-mentioned ferritin, and compares the activity stability with the natural enzyme. Utilizing the characteristics and methods of ferritin discovered by the present invention, the present invention can use ferritin from different species to mildly, conveniently, quickly, and in large quantities through biosynthesis or in vitro recombination to synthesize ferritin nanozymes with SOD-like enzyme activity, providing a material basis for the application of ferritin SOD nanozymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :a. SDS-PAGE of ferritin from different species before and after biosynthesis. b. Solution photos of ferritin from different species before and after biosynthesis. c. TEM electron microscope photos of ferritin from different species before and after biosynthesis. d. DLS particle size analysis of ferritin from different species before and after biosynthesis.

[0030] Figure 2:a. Detection of SOD enzyme activity before biosynthesis of ferritin from different species. b. Detection of SOD enzyme activity after biosynthesis of ferritin from different species. c. Detection of SOD enzyme activity of ferritin nanozymes containing concentration gradient iron in biosynthesis; d. Detection of activity of archaeal ferritin nanozymes before and after the removal of the iron core. e. Detection of SOD activity before and after the removal of the protein shell of archaeal ferritin nanozymes. f. SDS-PAGE detection before and after the removal of the protein shell of archaeal ferritin nanozymes. g. SDS-PAGE and protein solution photos of archaeal ferritin nanozymes before and after the removal of the iron core.

[0031] Figure 3 :a. Detection of iron content in ferritin nanozymes from different species. b. Detection of sulfur-iron ratio in ferritin nanozymes from different species. c. Detection of phosphorus-iron ratio in ferritin nanozymes from different species. d. SOD enzyme activity and phosphorus-iron ratio of human heavy chain ferritin nanozymes synthesized in vitro with gradient phosphorus-iron ratio. e.&f. SOD enzyme activity and phosphorus-iron ratio of archaeal pfFn and pyFn ferritin nanozymes synthesized in vitro with gradient phosphorus-iron ratio.

[0032] Figure 4 :a. Comparison of SOD activities of HFn, pfFn and pyFn ferritin nanozymes synthesized in vitro at the same iron-phosphorus ratio. b. Detection of phosphorus and iron content of HFn, pfFn and pyFn ferritin nanozymes synthesized in vitro at the same iron-phosphorus ratio. c. SOD enzyme activity of HFn, pfFn and pyFn ferritin nanozymes synthesized in vitro at the same iron-phosphorus ratio measured under conditions of equal iron content. d. Comparison of the electric potential of the triple axis of human heavy-chain ferritin and Pseudomonas aeruginosa ferritin. e. TEM electron microscopy of human heavy-chain ferritin mutants. f. Comparison of SOD enzyme activities of human heavy-chain ferritin mutants and wild type. g. Comparison of phosphorus-iron ratio of human heavy-chain ferritin mutants and wild type.

[0033] Figure 5 :a. High-resolution electron microscopy images of the core of human heavy chain ferritin and archaeal ferritin nanozyme, the scale bar represents 5 nm. b. Fourier transformed infrared spectrum of phosphate-doped 2-line ferrihydrite. c. X-ray diffraction pattern of phosphate-doped 2-line ferrihydrite. d. SOD enzyme activity detection of phosphate-doped 2-line ferrihydrite. e. High-resolution electron microscopy images and selected area diffraction images of phosphate-doped 2-line ferrihydrite.

[0034] Figure 6:a. SDS-PAGE electrophoresis of whole proteins of four transgenic strains before and after heat treatment. b. Growth curves of four transgenic strains before and after paraquat stimulation. c. Spot assay of four transgenic bacteria under paraquat stimulation. d. Photo of plate after paraquat stimulation of four transgenic strains. e. Bacterial viability detection of four transgenic strains before and after paraquat stimulation. f. SEM imaging of four transgenic strains before and after paraquat stimulation. g. Confocal imaging of live and dead bacteria staining of four transgenic strains before and after paraquat stimulation. h. Detection of ROS levels of four transgenic strains before and after paraquat stimulation.

[0035] Figure 7 :a. Activity unit detection of in vitro synthesized pyFn ferritin nanozyme. b. Initial SOD enzyme activity detection of ferritin nanozyme with equal activity and human CuZnSOD. c. SOD enzyme activity detection of ferritin nanozyme with equal activity and human CuZnSOD after 24h reaction. DETAILED DESCRIPTION

[0036] definition

[0037] Unless otherwise indicated, the terms used in the claims and specification are defined as shown below.

[0038] Unless otherwise defined herein, scientific and technical terms used in conjunction with the inventive methods and compositions described herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Typically, the nomenclature used in conjunction with the following and the following techniques are those well known and commonly used in the art: biochemistry, immunology, enzymology, molecular and cell biology, microbiology, genetics, and polypeptide chemistry as described herein.

[0039] Unless otherwise indicated, the methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry: Section A Proteins, Vol. I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol. II, CRC Press (1976).

[0040] All publications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.

[0041] Unless otherwise indicated, the following terms shall be understood to have the following meanings.

[0042] When used in conjunction with a number, the term "about" refers to any number that is within ±1, ±5, ±10%, or ±15% of the referenced number.

[0043] The term "nanozyme" refers to a class of nanomaterials that have enzymatic properties. They can catalyze enzyme substrates, produce catalytic reactions similar to natural enzymes, and have characteristics such as enzymatic reaction kinetics. They are a new type of simulated enzyme.

[0044] The term "nanozymes with SOD activity" refers to a class of nanomaterials that contain SOD enzymatic properties and can catalyze the release of superoxide anion free radicals (O2 -) is dismutated into H2O2 and O2, producing a catalytic reaction similar to that of the natural SOD enzyme, and has the characteristics of SOD enzymatic reaction kinetics, belonging to a new type of mimetic enzyme.

[0045] The term "ferritin nanozyme" refers to an inorganic nanoparticle with enzyme-like activity synthesized in the lumen of ferritin. In some embodiments, the ferritin nanozyme consists of a ferritin shell and a core with nanozyme activity therein. In some embodiments, the ferritin nanozyme is only a core with nanozyme activity, and does not include a ferritin shell. In some embodiments, the ferritin shell is removed by protease degradation.

[0046] The term "ferritin nanozyme with SOD activity" refers to a core with SOD activity synthesized in situ by the iron oxidase center and nucleation site of ferritin itself. In some embodiments, the ferritin nanozyme with SOD activity consists of a ferritin shell and a core with SOD activity therein. In some embodiments, the ferritin nanozyme with SOD activity is only a core with SOD activity, and does not include a ferritin shell. In some embodiments, the ferritin shell is removed by protease degradation.

[0047] In some embodiments, the ferritin of the present invention may be ferritin of natural origin, or may be recombinantly expressed ferritin, or a mutant thereof, which may be derived from prokaryotes, protists, fungi, plants or animals, such as archaea, bacteria, fungi, insects, reptiles, birds, amphibians, fish, mammals, such as rodents, ruminants, non-human primates or humans, such as mice, rats, guinea pigs, dogs, cats, cattle, horses, sheep, monkeys, gorillas, and humans. From archaea, bacteria to humans, although the amino acid sequences of ferritin in different organisms are very different, their structures are similar and can form a protein shell structure.

[0048] The term "human H ferritin" refers to ferritin formed by self-assembly of human ferritin H subunits.

[0049] In some embodiments, the ferritin nanozyme with SOD enzyme activity in the present invention is derived from an archaea (also known as archaea), which is selected from Crenarchaeota., Euryarchaeota., Nanoarchaeota. or Thaumarchaeota. In some embodiments, the archaea is selected from Thermoprotei, Caldisphaerales, Caldisphaeraceae, Caldisphaera, Desulfurococcales, Desulfurococcaceae, Acidilobus, Aeropyrum, Desulfurococcus, Igni coccus, Ignisphaera, Staphylothermus, Stetteria, Sulfophobococcus, Thermodiscus, Thermosphaera, Pyrodictiaceae, Hyperthermus, Pyrodictium, Pyrolobus, Sulfololobales bales), Sulfolobaceae, Acidianus, Desulfurolobus, Metallosphaera, Stygiolobus, Sulfolobus, Sulfurisphaera, Sulfurococcus, Thermoproteales, Thermofilaceae, Thermofilaceae, Thermofilum), Thermoproteaceae, Caldivirga, Pyrobaculum, Thermocladium, Thermoproteus, Vulcanisaeta, Archaeoglobi, Archaeoglobales, Archaeoglobaceae, Archaeoglobus,Ferroglobus, Geoglobus, Halobacteria, Halobacteriales, Halobacteriaceae, Haladaptatus, Halalkalicoccus, Haloarcula, Halobacterium, Halobaculum, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halomicrobium, Halopiger, Haloplanus, Haloquadratum, Halorhabdus, Halorubrum, Halosimplex, Halostagnicola, Haloterrigena, Halovivax, Natrialba, Natrinema, Natribacillum atronobacterium, Natronococcus, Natronolimnobius, Natronomonas, Natronorubrum, Methanobacteria, Methanobacteriales, Methanobacteriaceae, Methanobacterium, Methanobrevibacter, Methanospore haera), Methanothermobacter, Methanothermaceae, Methanothermus, Methanococci, Methanococcales, Methanocaldococcaceae, Methanocaldococcus, Methanotorris, Methanococcaceae, Methanococcus,Methanothermococcus, Methanomicrobia, Methanomirobiales, Methanocorpusculaceae, Methanocorpusculum, Methanomicrobiaceae, Methanoculleus, Methanofollis, Methanogenium, Methanolacinia, Methanobacterium Methanomicrobium, Methanoplanus, Methanospirillaceae, Methanospirillum, Methanopyri, Methanopyrales, Methanopyraceae, Methanopyrus, Thermococci, Thermococcales, Thermococcaceae, Palaeococcaceae ccus), Pyrococcus, Thermococcus, Thermoplasmata, Thermoplasmatales, Ferroplasmataceae, Ferroplasma, Picrophilaceae, Picrophilus, Thermoplasmataceae, Thermoplasma, Methanocalculus, Methanoli nea), Methanosarcinales, Methanosaetaceae, Methanosaeta, Methanothrix, Methanosarcinaceae, Halomethanococcus, Methanimicrococcus, Methanococcoides, Methanohalobium, Methanohalophilus,Methanolobus, Methanomethylovorans, Methanosalsum, Methanosarcina, Methermicoccaceae, Methermicoccus, Thermogymnomonas, Korarchaeum, Nanoarchaeum, Cenarchaeales, Cenarchaeaceae, Cenarchaeum, Nitrosocaldales, Nitrosocaldaceae, Nitrosocaldus, Nitrosopumilales, Nitrosopumilaceae or Nitrosopumilus. In some embodiments, the archaea is selected from the Sulfolobales. In further embodiments, the archaea is selected from the Sulfolobaceae. In further embodiments, the archaea is selected from the genus Sulfolobus. In some embodiments, the archaea is selected from the genus Pyrococcus.

[0050] In some embodiments, the ferritin nanozyme with SOD enzyme activity in the present invention is derived from bacteria. In some embodiments, the bacteria are selected from cocci, bacilli or spiral bacteria (including vibrio, spirilla, helicobacter). In some embodiments, the bacteria are selected from bacilli, such as Escherichia coli or Pseudomonas aeruginosa.

[0051] In some embodiments, the ferritin nanozyme of the SOD enzyme activity of the present invention is derived from fungi. In some embodiments, the fungi are selected from Chytridiomycota, Zygomycota, Ascomycota or Basidiomycota. In some embodiments, the fungi are selected from Ascomycetes, Taphrinomycetes, Basidiomycetes, Urediniomycetes, Ustilaginomycetes, Zygomycetes, Trichomycetes.

[0052] The culture method of the archaea and bacteria of the present invention is not particularly limited, and the culture can be carried out according to the corresponding culture method of the corresponding bacteria.

[0053] In some embodiments, the buffer in step 2) of method 1 of the present invention can be 5-100 mM Tris buffer at pH 7.5-8.5 or PBS buffer at pH 7.0-8.0.

[0054] In some embodiments, the bacteria of method 1 can be disrupted by high-pressure disruptor, repeated freezing and thawing, ultrasonic disruption, or osmotic pressure shock.

[0055] In some embodiments, the conditions for removing insoluble matter and cell debris by centrifugation in method 1 are 10,000-18,000 x g for 10-50 min, such as about 12,000 x g for 30 min.

[0056] In some embodiments, the cooling of method 1 is achieved using an ice bath.

[0057] In some embodiments, the anion purification column of Method 1 is Q Sepharose Fast Flow (GE Health).

[0058] In some embodiments, the molecular sieve of Method 1 is Superdex 200 prep grade (GE Health).

[0059] In some embodiments, the ferritin shell can be removed in method 1 by digesting with proteases, depolymerizing the ferritin shell with high temperature or urea or guanidine hydrochloride, and then centrifuging, such as digesting with proteinase K, depolymerizing with 8M urea, and then centrifuging at about 12000 x g for 5-30 min.

[0060] In some embodiments, the rapid mixing of method 2 can be achieved by manual stirring or magnetic stirring, such as magnetic stirring at about 220 rpm.

[0061] In some embodiments, the concentration of the aqueous phosphate solution in methods 2 and 3 is 0.001-0.5 M, preferably, 0.002-0.2 M, more preferably, 0.005-0.1 M, more preferably, 0.02-0.07 M. In some embodiments, the concentration is the final concentration after addition.

[0062] In some embodiments, the phosphate is a sodium, potassium, or ammonium phosphate, such as dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate.

[0063] In some embodiments, the rapid addition of the aqueous solution of iron salt in method 2 means that for a 0.25L reaction system, the solution is added within 5 seconds to 5 minutes, such as 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes. In some embodiments, the iron salt of method 2-3 is ferric nitrate. In some embodiments, the concentration of the iron salt of method 2-3 is 0.01-0.5M, such as 0.02-0.2M, preferably, 0.05-0.1M. In some embodiments, this concentration is the final concentration after addition.

[0064] In some embodiments, the slow addition of NaOH or KOH solution in method 2 means that for a 0.5L reaction system, the alkali solution is added within 30 seconds to 120 minutes, such as within 45 seconds, 60 seconds, 90 seconds, 120 seconds, 3 minutes, 5 minutes, 10 minutes, 20 minutes, 40, 60, 80, 100 minutes. In some embodiments, the concentration of NaOH or KOH is 0.1-3M, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5M. In some embodiments, the temperature of the reaction in step b) of method 2 is 10-40°C, such as 15-35, 20-30°C, such as room temperature (about 25°C). In some embodiments, the reaction lasts for 15 min-1.5 hr, such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 70 min, 80 min. In some embodiments, the pH in step b) of method 2 is adjusted to near neutrality, such as pH 6.5-7.5, 6.8-7.2. In some embodiments, the reaction of step b) of method 2 is carried out for 15 min-1.5 hr, such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 70 min, 80 min.

[0065] In some embodiments, the centrifugation conditions of method 2 are 3000-10000 x g for 5-30 min and washing 2-10 times.

[0066] In some embodiments, the slowly adding of the ferrous salt solution in step iii) of method 3 means that for a reaction system of 1-500 mL, the ferrous salt solution is added within 30 seconds to 120 minutes, such as within 45 seconds, 60 seconds, 90 seconds, 120 seconds, 3 minutes, 5 minutes, 10 minutes, 20 minutes, 40, 60, 80, 100 minutes. In some embodiments, the ferrous salt solution is slowly added in batches, such as each time at a ratio of 100-1000:1 (iron ion: ferritin), 2-30 minutes / time, and a total of 4-10 times of adding the ferrous salt.

[0067] In some embodiments, the ferrous salt and phosphate added in method 3 are ammonium ferrous sulfate and dipotassium hydrogen phosphate, respectively, and the amount added each time is 50-1500 moles of iron or phosphorus per mole of ferritin, preferably 100-1000, 200-800, 300-500, and the addition time interval is 5-20 min, preferably 8-15 min.

[0068] In some embodiments, the reaction temperature of method 3 is 15-40°C, preferably, 16-37°C, more preferably, 20-30°C, even more preferably, 23-27°C, such as about 25°C.

[0069] In some embodiments, the centrifugation condition of method 3 is 8000-20000 x g for 5-30 min. In some embodiments, desalting is performed by means of a desalting column.

[0070] In some embodiments, the recombinant cell of method 4 can be a bacterium or fungus available in the art for recombinant expression of recombinant proteins. In some embodiments, the recombinant cell is Escherichia coli, yeast, preferably Escherichia coli.

[0071] In some embodiments, the inducible expression method of method 4 can be environmental induction or inducer-inducible expression available in the art, such as temperature-inducible expression, methanol-inducible expression or IPTG-inducible expression.

[0072] In some embodiments, the ferrous salt of Method 4 can be ammonium ferrous sulfate, ferrous chloride, ferrous nitrate, or ferrous sulfate.

[0073] In some embodiments, the concentration of the aqueous ferrous salt solution of method 4 is 1-25 mM, preferably, 2-20 mM, more preferably, 3-15 mM, more preferably, 5-10 mM.

[0074] In some embodiments, the culture temperature after adding the ferrous salt aqueous solution in method 4 is 23-37°C, preferably, 25-35°C, and more preferably, 28-32°C.

[0075] In some embodiments, the culture time after adding the ferrous salt aqueous solution in method 4 is 5-15 hr, preferably, 8-10 hr.

[0076] In some embodiments, steps IV) and V) of method 4 are similar to steps 2)-4) of method 1.

[0077] In some embodiments, the isolated nanozyme with SOD activity of the present invention is provided in the form of a solution or a lyophilized powder. In some embodiments, the isolated nanozyme with SOD activity of the present invention is provided in the form of a composition, such as a pharmaceutical composition comprising an excipient.

[0078] In some embodiments, the isolated nanozyme with SOD activity of the present invention can be used as a scavenger of superoxide free radicals in the subject's body, and is used to prevent and / or treat various conditions and / or diseases caused by superoxide free radicals in the body, such as delaying human aging, improving human immunity, preventing and / or treating myocardial infarction, atherosclerosis, diabetes, vascular heart disease, cancer, arthritis, fatty liver, cirrhosis, renal failure, gastrointestinal ulcers, cataracts, collagen diseases, neonatal dyspnea syndrome, edema, emphysema, oxygen poisoning, psoriasis, dermatitis, eczema, pruritus, radiation sickness, etc.

[0079] In some embodiments, the subject is a rodent, farm animal, companion animal, non-human primate, or human, such as a mouse, rat, guinea pig, rabbit, sheep, cow, horse, donkey, pig, cat, dog, monkey, gorilla, etc.

[0080] In some embodiments, the ferritin nanozyme with SOD enzyme activity of the present invention can be added to food as a natural antioxidant to act as a preservative.

[0081] In some embodiments, the ferritin nanozyme with SOD enzyme activity of the present invention can be used in the daily chemical industry, such as as a skin care product and toothpaste to prevent skin aging, reduce wrinkles and remove spots, achieve skin care effects, or prevent and treat related skin diseases, such as dermatitis, acne, skin burns, etc., and plays an important role in oral and dental health care, such as for preventing and treating periodontitis.

[0082] In some embodiments, the ferritin nanozyme with SOD enzyme activity of the present invention can be used to enhance the stress resistance of microorganisms or plants. By overexpressing the ferritin nanozyme with SOD enzyme activity of the present invention in transgenic microorganisms or plants, the resistance of microorganisms or plants to harsh environments can be improved, and the tolerance of transgenic microorganisms or plants to oxygen stress can be improved.

[0083] The present invention is further described below in conjunction with specific implementation cases, and the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The reagents used in the following examples, if not specifically noted, can be easily purchased from reagent companies such as Sigma Aldrich, Merck, and the test methods, if not specifically noted, can be found in textbooks such as Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York. For those skilled in the art, any changes, modifications made within the scope of the present invention or the examples implemented by directly adopting the equivalent conditions in the embodiments, should be understood as being within the scope of the present invention.

[0084] Example

[0085] Example 1 Synthesis and characterization of ferritin nanozymes from different species

[0086] method:

[0087] Construction of ferritin expression plasmids from different species

[0088] In order to explore the enzyme-like activity of ferritin nanozymes from different species, the present invention selected sequences covering three major species and three ferritin structures, including Dps from archaea: Sulfolobus solfataricus and bacteria: Escherichia coli (SEQ ID NO.1&2, the first 6 nucleotides are restriction sites, the last nine nucleotides are stop codons and restriction sites), Bfr from bacteria: Escherichia coli (SEQ ID NO.3, the first 6 nucleotides are restriction sites, the last nine nucleotides are stop codons and restriction sites) and from bacteria: Escherichia coli (SEQ ID NO.4, the first 6 nucleotides are restriction sites, the last nine nucleotides are stop codons and restriction sites), archaea: Pyrococcus furiosus and Pyrococcus yayanosii (SEQ ID 5&6, the first 6 nucleotides are restriction sites, the last nine nucleotides are stop codons and restriction sites) and FTn from eukaryotic organisms: human heavy chain ferritin, light chain ferritin, mitochondrial ferritin (SEQ ID NOs.7-9, the first 6 nucleotides are restriction sites, the last nine nucleotides are stop codons and restriction sites) and human light and heavy chain heteromeric ferritin (SEQ ID NOs.10&11) from Homo sapiens (SEQ ID NOs.1-9) were cloned into the E. coli expression vector pet22b(+) using NdeI and BamHI restriction enzyme sites, and SEQ ID NOs.10&11 were cloned into the two multiple cloning sites of petDuet-1 using NcoI and BamHI, NdeI and XhoI, respectively, and the sequences were confirmed to be correct by DNA sequencing.

[0089] Biosynthesis of ferritin nanozymes from different species

[0090] The plasmid obtained above was transferred into the E. coli expression strain BL21 (DE3), and the transformed strain was cultured overnight in LB medium containing 100 mg / L ampicillin, and then 1 mM IPTG (Sigma-Aldrich) and 1-10 mM freshly prepared ammonium ferrous sulfate solution (Sigma-Aldrich) were added simultaneously, and induced expression at 30°C and 220 rpm for 8 hours. During this process, excess ferrous ions will enter the ferritin lumen and be oxidized into nuclei.

[0091] Protein purification: The induced culture was centrifuged at 4000x g for 15 min to collect the cells, and the cells were resuspended in 20mM Tris buffer at pH 8.0. The resuspended cells were broken by a high-pressure crusher. The broken cells were centrifuged at 12000x g for 30 min to collect the supernatant for subsequent purification. Different purification strategies were used for ferritin from different species: the Dps supernatant of S. solfataricus was in a 65°C water bath for 10 min, placed on ice, and then centrifuged at 12000x g for 30 min. The supernatant was collected and removed by anion exchange column Q-Sepharose Fast Flow (GE Healthcare) to remove impurities, and further purified by Superdex 200 (16 / 200GL, GE Healthcare) molecular sieve. The Dps supernatant of E. coli was directly purified by anion purification column to remove most impurities and nucleic acids, and then further purified by cation purification column (SP Sepharose Fast Flow, GE Healthcare) and Superdex 200 molecular sieve. The supernatant of Bfr of E.coli was placed in a 65℃ water bath for 10 min, cooled on ice and centrifuged at 12000x g for 30 min, and the supernatant was purified using anion purification columns and molecular sieves. Ferritin of P.furiosus and P.yayanosii, human heavy chain and light chain ferritin, human mitochondrial ferritin, and human light and heavy chain heteromeric ferritin were all placed in a 72℃ water bath for 15 min, cooled on ice and the supernatant was purified using anion purification columns and molecular sieves. The protein concentration of the purified protein was determined using a BCA protein quantification kit (Pierce), and the enzyme activity was determined using a SOD enzyme activity detection kit (Dojindo).

[0092] result:

[0093] The results of SDS-PAGE showed that ferritin from 11 different sources could be expressed and purified by biosynthesis, and the size of the protein monomer did not change significantly before and after biosynthesis ( Figure 1 TEM images and DLS particle size analysis results show that the biosynthesis process has no significant effect on the structure of ferritin from different sources ( Figure 1 c and d). In E. coli Bfr and FTn, human light and heavy chain heteromeric ferritin (H / LFn), human mitochondrial ferritin (MFn), human heavy chain ferritin (HFn), and P. furiosus and P. yayanosii ferritin (pfFn, pyFn), the biosynthesized protein solution showed a distinct reddish brown solution ( Figure 1 At the same time, the electron microscopy photos without negative staining showed that a significant inner core was formed in the lumen of different ferritins ( Figure 1c). However, since there is no iron oxidase center in the inner cavity of human light chain ferritin and the Dps ferritin is smaller in size, the iron core they form is smaller in size and the color change of the protein solution is not obvious.

[0094] Example 2 Detection of SOD enzyme activity of biosynthetic ferritin nanozyme and verification of its activity source

[0095] method:

[0096] 1. Removal of iron core of biosynthetic ferritin nanozyme: The ferritin nanozyme obtained above at a final concentration of 1 mg / mL was added to a 0.1 M sodium acetate solution containing 1% thioglycolic acid, pH 5.5, and slowly rotated at 4°C for 18 h. Then, an excess of bipyridine (Sigma-Aldrich) relative to iron was added, and the treated solution was passed through a desalting column (Superdex 75 prepgrade, GE Health) to separate ferritin and free iron.

[0097] 2. Ferritin protein shell degradation: The ferritin solution obtained above with a final concentration of 1 mg / mL was added to a 20 mM Tris buffer at pH 8.0 containing an equal amount of proteinase K (Lambolide). The mixed solution was placed in a 55°C water bath for 24 h to fully degrade the protein shell. The treated solution was used for SDS-PAGE detection and SOD enzyme activity determination.

[0098] result:

[0099] After testing the SOD enzyme activity of ferritin nanozymes from different sources of biosynthesis, we found that the SOD enzyme activity of ferritin nanozymes showed a significant species correlation: prokaryotes (archaea, bacteria) > eukaryotes ( Figure 2 At the same time, by adding gradient ferrous salts during the biosynthesis process, we found that the SOD enzyme activity of ferritin nanozymes was significantly correlated with its iron content ( Figure 2 (c).

[0100] After using 1% thioglycolic acid and excess bipyridine to reduce and remove the ferritin core, the photo of the ferritin solution showed that the iron core was significantly removed, while the SDS-PAGE results showed that the ferritin structure did not change significantly ( Figure 2 g). After measuring the activity of ferritin nanozymes before and after treatment, it can be seen that the SOD enzyme activity is significantly reduced, indicating that the activity of biosynthetic ferritin nanozymes comes from its core ( Figure 2 d). Further, by adding proteinase K to the ferritin solution and incubating at 55°C for 24 h, SDS-PAGE results showed that the ferritin shell was significantly degraded ( Figure 2 f), but its activity is still largely retained ( Figure 2Figure e), further illustrating that the SOD enzyme activity of ferritin nanozyme originates from the inner core.

[0101] Example 3 The difference in activity of ferritin nanozymes from different species is due to the different ratios of iron and phosphorus in the inner core

[0102] method:

[0103] Synthesis of recombinant ferritin nanozyme in vitro; wild-type ferritin from different species with a final concentration of 1 mg / mL was added to 50 mM MES buffer at pH 6.5, and fresh deionized water was used to prepare equimolar concentrations of ammonium ferrous sulfate and potassium dihydrogen phosphate mother solutions. When synthesizing a single iron-containing core, ammonium ferrous sulfate solution was slowly added to the ferritin solution at a ratio of 500:1 (iron ions: ferritin) at a frequency of 10 min / time, for a total of 6-7 times, and the iron oxidase center and nucleation site in the ferritin lumen were used to synthesize ferritin nanozyme in vitro. When synthesizing ferritin nanozyme containing a gradient phosphate core, a corresponding proportion of potassium dihydrogen phosphate solution was added before adding the ammonium ferrous sulfate solution. After the synthesis, the precipitate was removed by centrifugation at 12000 x g for 10 min, and the supernatant was purified with a desalting column.

[0104] result:

[0105] 1. The main elements (iron, phosphorus, sulfur) content of ferritin nanozymes with different SOD enzyme activities was detected (ICP / MS). The results showed that the SOD enzyme activity from different species did not show a significant correlation with the iron content and the iron-sulfur ratio, but was significantly positively correlated with the phosphorus-iron ratio. This preliminarily indicates that the difference in the activity of ferritin nanozymes from different species is derived from the difference in the phosphorus and iron content of their cores ( Figure 3 a, b and c).

[0106] 2. After synthesizing ferritin nanozymes containing gradient iron-phosphorus ratios in vitro, we found that the activity of ferritin nanozymes was significantly affected by the iron-phosphorus ratio added to them. The higher the phosphorus / iron ratio, the stronger its ability to remove superoxide anions ( Figure 3 d, e and f).

[0107] Example 4 Verification that the structure of ferritin determines its inner core composition

[0108] method:

[0109] 1. Using Pseudomonas aeruginosa ferritin (NCBI accession number: WP_003092078.1) as a reference, the amino acids on the triple axis of human heavy chain ferritin were mutated to the amino acids at the corresponding positions in Pseudomonas aeruginosa (T123E, C131R, E135K, T136D) (SEQ ID NO.12, the first 6 nucleotides are restriction sites, and the last nine nucleotides are stop codons and restriction sites). The mutated amino acid sequence was synthesized by whole gene, cloned into the Escherichia coli expression vector pet22b (+) using NdeI and BamHI restriction enzymes, and confirmed to be correct by DNA sequencing. The plasmid obtained above was transferred into the Escherichia coli BL21 (DE3) expression strain, and the transformed Escherichia coli was grown overnight in LB medium containing 100 mg / L ampicillin, and then 1 mM IPTG was used to culture at 30°C for 8 hours to induce protein expression. The protein purification process was the same as that of wild-type HFn.

[0110] result:

[0111] 1. Since ferritins from different sources show different iron-phosphorus content and SOD enzyme activity under the same synthesis conditions, in order to verify the influence of protein structure on the core composition, the present invention selects HFn, pfFn and pyFn with large differences in activity as research objects, and synthesizes nanozymes in vitro under the same iron-phosphorus ratio. The results show that the activity and phosphorus-iron ratio of human heavy chain ferritin nanozymes under the same conditions are significantly lower than those of archaeal ferritin nanozymes, and the activity of human heavy chain ferritin nanozymes is still significantly lower after detection under the same iron content, indicating that different core compositions and SOD enzyme activities are determined by protein structure ( Figure 4 a, b and c).

[0112] 2. In order to further verify the decisive role of protein structure, the present invention mutated human heavy chain ferritin to improve its affinity for phosphate. To this end, the present invention selected Pseudomonas aeruginosa ferritin as the object, and found the presence of sulfate ions in the triple axis of iron-soaked crystals, indicating that it may serve as an internalization site for anions. At the same time, the potential analysis of the triple axis also showed that compared with human heavy chain ferritin, the electronegativity of the triple axis of Pseudomonas aeruginosa ferritin was weaker, which was more conducive to the internalization of anions ( Figure 4 Human heavy chain ferritin mutants were characterized and their protein structure did not change significantly ( Figure 4 e figure), and after measuring its activity and phosphorus and iron content, we found that the mutation can significantly improve the phosphate uptake capacity and SOD enzyme activity of human heavy chain ferritin ( Figure 4 f&g diagram).

[0113] Example 5 Identification of the Effect of Phosphate on Ferritin Core Structure and Activity

[0114] method:

[0115] 1. Synthesis of phosphate-doped 2-line ferrihydrite: Existing work has shown that the ferritin core is a ferrihydrite-like structure (reference 1). In order to detect the effect of phosphate on the ferritin core, the present invention simulates the ferritin core of different biological sources by incorporating dipotassium hydrogen phosphate with a concentration gradient during the ferrihydrite synthesis process. 250mL of 0.1M, 0.05M, 0.02M, and 0.01M K2HPO4 aqueous solutions were added to a 1L beaker in advance, and 250mL of 0.1M Fe(NO3)3 was quickly added under rapid mixing conditions, followed by slow addition of 1M KOH to adjust the solution pH to about 7, and the reaction was performed at room temperature for 30min to synthesize 2-line ferrihydrite doped with different phosphoric acids. The synthesized phosphate ferrihydrite was centrifuged and washed 5 times at 5000x g and freeze-dried for standby use.

[0116] 2. Characterization of ferritin nanozymes and phosphate-doped 2-line ferrihydrite: The lattice morphology and selected diffraction of ferritin nanozymes and phosphate-doped ferrihydrite were characterized by field emission transmission electron microscopy (FEITecnai G2 F30, USA). The infrared spectrometer (Nicolet is10, USA) was used in the wavelength range of 400-4000cm -1 4cm inside -1 The functional groups of ferrihydrite were characterized with a resolution of 1.3 Å. The crystal structure of ferrihydrite was characterized using an X-ray powder diffractometer (Brucker D8 Advance, Germany) with a copper target in the range of 10-90° at a scanning rate of 2° / min.

[0117] result:

[0118] 1. High-resolution electron microscopy was used to characterize the human heavy-chain ferritin nanozymes and archaeal ferritin nanozymes with significantly different activities. The results showed that the core of human heavy-chain ferritin showed a single crystal phenotype, and when phosphate was recombined into the ferritin core, the core of human heavy-chain ferritin transformed from a single crystal to a polycrystalline one. In the archaeal pfFn and pyFn ferritin nanozymes with higher phosphate content, their cores were completely amorphous in morphology, indicating that phosphate can significantly affect the structure of the ferritin core ( Figure 5 (a).

[0119] 2. In order to further verify the effect of phosphate on the core structure and activity, the present invention directly synthesized gradient phosphate-doped 2-line ferrihydrite in vitro. The Fourier transform infrared spectrum FTIR at 1060 cm -1 The characteristic peaks that appeared around 100% and increased significantly with the addition of phosphate proved the successful incorporation of phosphate ( Figure 5At the same time, the XRD diffraction pattern shows that with the addition of phosphate, the second peak in the diffraction pattern of 2-line ferrihydrite is significantly reduced, indicating that the addition of phosphate can significantly change the crystal structure of 2-line ferrihydrite ( Figure 5 c). In the results of high-resolution electron microscopy and electron selected area diffraction, the incorporation of phosphate caused the 2-line ferrihydrite to transform from single crystal to amorphous and the diffraction points in the selected area diffraction to disappear significantly, showing a structure similar to the ferritin core. More importantly, the doping of phosphate can significantly increase the SOD enzyme activity of 2-line ferrihydrite, further illustrating the effect of phosphate on the structure and activity of the core ( Figure 5 d and e).

[0120] Example 6 Verification of the antioxidant physiological function of ferritin SOD nanozyme

[0121] method:

[0122] 1. Antioxidant growth curve determination: After cloning the HFn, pfFn and pyFn expression sequences into the pet22b(+) vector, 100ng of the above three plasmids and the empty vector were transformed into 100μL of E. coli BL21(DE3) competent cells, and spread on LB plates containing 100mg / L ampicillin, and cultured at 37℃ overnight. After forming a single clone, pick the single clone and place it in a fresh LB medium containing ampicillin. After overnight culture, adjust the OD600 of the four transgenic bacteria to the same level and transfer them to fresh culture medium. When the OD600 reaches 0.2, add IPTG with a final concentration of 1mM and superoxide anion generator paraquat (Macklin) at 1mM at the same time, and place it at 37℃, 220rpm for 12h, record the OD600 reading every two hours, and repeat each sample 3 times.

[0123] 2. Bacterial activity detection: The bacterial activity was detected using a bacterial activity detection kit (Dojindo). After the four strains were treated according to the above method, 190 μL of bacterial solution was taken to a 96-well plate and 10 μL of the WST dye provided by the kit (9 μL of WST solution + 1 μL of electron carrier solution) was added to each well. After mixing, the OD450 reading was detected after placing at 37°C for 2-4 hours.

[0124] 3. Bacterial live and dead staining: The paraquat-treated bacterial solution was detected using the ThermoFisher Bacterial Live and Dead Staining Kit (ThermoFisher, USA). 1 mL of the treated bacteria was collected by centrifugation at 10000 x g for 10 min and washed twice with saline. 1 μL of SYTO-9 and PI dyes in the kit were added to each mL of bacterial solution, incubated at room temperature in the dark for 15 min, and 5 μL of the bacterial solution was dropped onto a glass slide for observation under a confocal microscope.

[0125] 4. Detection of ROS in bacteria: Take 1 mL of the above four unstimulated and stimulated bacteria, wash twice with saline, add DCFH-DA solution (Sigma-Aldrich) containing a final concentration of 10 μM, and incubate at 37°C in the dark for 30 minutes. Then wash twice with saline and resuspend in 1 mL of saline, and detect ROS levels using flow cytometry.

[0126] 5. Spot assay: Transfer the four transgenic bacteria cultured overnight to fresh culture medium, and add 1mM IPTG to induce expression for 8h when the OD600 reaches 0.6-0.8. Then dilute the four bacterial solutions to the same OD600 level with LB culture medium, and add them dropwise to the culture medium containing 0.2mM paraquat and 100mg / L ampicillin after 10-fold gradient dilution. After culturing at 37℃ overnight, take photos and record.

[0127] result:

[0128] The HFn sequence with low SOD enzyme activity and the archaeal ferritin pfFn and pyFn sequences with high SOD enzyme activity were cloned into the pet22b(+) expression vector. Then, equal amounts of the empty vector and the three plasmids containing the coding sequences were transformed into competent cells of Escherichia coli BL21(DE3). SDS-PAGE results showed that the three proteins were all expressed in the expression strain and the expression levels were similar ( Figure 6 Figure a). Subsequently, by testing the growth curves of the four strains under paraquat stimulation, it can be seen that the pfFn and pyFn groups with high SOD enzyme activity have a more obvious growth advantage under paraquat stimulation ( Figure 6 (Figure b). At the same time, the four strains after induced expression were gradient diluted and added to the plate containing 0.2mM paraquat and ampicillin. The results also showed that the three ferritins transferred into the plate can significantly improve the tolerance of bacteria to superoxide anions. The pfFn and pyFn groups with high SOD enzyme activity had a more obvious mitigation effect, which was also verified in the re-plating experiment after paraquat stimulation and the bacterial activity test ( Figure 6 d and e). At the same time, the bacterial morphology before and after stimulation was characterized by scanning electron microscopy (SEM). It can be seen that the four bacteria before treatment all showed a relatively full rod-shaped structure, while the bacteria in the empty group showed obvious shrinkage after paraquat stimulation. The bacteria transferred to HFn could alleviate the shrinkage to a certain extent, but there was still some shrinkage. After the bacteria were transferred to pfFn and pyFn with high SOD enzyme activity, the bacteria maintained an obvious saturated rod-shaped structure, proving the antioxidant effect of archaeal ferritin nanozyme ( Figure 6f), and the confocal photos of bacterial live and dead staining further proved that there were more live bacteria in the pfFn and pyFn groups. At the same time, by detecting the level of ROS in bacteria before and after stimulation, we also found that the introduction of archaeal ferritin can significantly inhibit the ROS produced by paraquat, further demonstrating its ability to degrade superoxide anions ( Figure 6 h figure).

[0129] Example 7 Synthesis of Ferritin SOD Nanozyme and Comparison with Natural SOD Enzyme

[0130] Based on the active source of ferritin SOD nanozyme discovered by the present invention, the present invention uses highly active archaeal ferritin pyFn to directly synthesize ferritin nanozyme in vitro. After the activity of the synthesized ferritin nanozyme was tested, the results showed that its SOD enzyme activity reached nearly 3500U / mg ( Figure 7 Figure a). Based on the excellent thermal stability of archaeal ferritin, the present invention compares archaeal ferritin SOD nanozyme and human CuZnSOD with equal enzyme activity, and finds that archaeal ferritin nanozyme has a longer-lasting effect of removing superoxide anions than natural enzymes, which illustrates the good application prospects of ferritin SOD nanozyme ( Figure 7 b and c).

[0131] Equivalent solution

[0132] Although multiple embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily envision various other means and / or structures for achieving the functions described herein and / or obtaining the results and / or one or more advantages described herein, and it is believed that each such change and / or modification is within the scope of the present invention. More broadly, it will be readily understood by those skilled in the art that all parameters, materials and settings described herein are intended to be exemplary, and that actual parameters, materials and / or settings will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments and examples are presented only by way of example, and within the scope of the appended claims and their equivalents, the present invention may be implemented in a manner different from that specifically described and claimed. Any combination of two or more such features, systems, articles, materials and / or methods is included within the scope of the present invention if such features, systems, articles, materials and / or methods are not mutually conflicting.

[0133] The phrase "and / or" as used in the present specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and not in other cases. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to A but not B (optionally including other elements in addition to B) in one embodiment; to B but not A (optionally including elements in addition to A) in another embodiment; to both A and B (optionally including other elements) in yet another embodiment; and so on.

[0134] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be understood to be inclusive, i.e., including at least one of a plurality of elements or lists of elements, but also including more than one, and optionally, other unlisted items. Only when the contrary term is explicitly indicated, such as "only one of" or "exactly one of", or when used in the claims, "consisting of" will refer to exactly one element of a plurality of elements or lists of elements. Generally, when preceded by an exclusive term, such as "either", "one of", "only one of" or "exactly one of", the term "or" used herein should be understood only to represent an exclusive alternative (i.e., "one or the other but not both"). "Substantially consisting of" when used in the claims shall have its ordinary meaning in the field of patent law.

[0135] As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements of the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than the elements specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") may refer to at least one, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0136] In the claims and the above description, all conjunctions, such as "comprises", "includes", "with", "having", "containing", "involving", "having", etc., are understood to be open-ended, i.e., meaning including but not limited to. Only the conjunctions "consisting of" and "consisting essentially of" shall be closed or semi-closed conjunctions, respectively.

[0137] The use of ordinal terms in the claims, such as "first", "second", "third", etc. to modify claim elements does not itself imply any priority, precedence or order of one claim element relative to another claim element, or the temporal order of actions in a method, but merely serves as a label to distinguish one claim element with a certain name from another element with the same name (but used in ordinal terms) to distinguish claim elements.

[0138] References:

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Claims

1. An isolated nanozyme with SOD activity, which is ferrihydrite containing phosphorus.

2. The isolated nanozyme with SOD activity as claimed in claim 1, which is isolated from ferritin of archaea or bacteria, obtained by utilizing the ferrihydrite synthesis process, or synthesized in situ by adding ferrous salt during the expression of ferritin using the oxidase center and nucleation site of ferritin itself, or recombinantly synthesized in vitro by adding ferrous salt and phosphate to the solution system after the ferritin is purified, and it is a nanozyme with SOD activity contained in the ferritin shell or a nanozyme with SOD activity that does not contain a ferritin shell, wherein the isolated nanozyme with SOD activity is obtained by incorporating phosphate during the ferrihydrite synthesis process or by adding phosphate during the expression of ferritin or in the solution system.

3. The isolated nanozyme with SOD activity as described in claim 2, wherein the iron salt is a ferrous salt, and more preferably, the metal salt is a ferrous sulfate salt.

4. The isolated nanozyme with SOD activity as described in claim 2 or 3, wherein the phosphate is potassium, sodium or ammonium phosphate, more preferably, the phosphate is potassium phosphate.

5. The isolated nanozyme with SOD activity as described in any one of claims 2 to 4, wherein the ferritin is bacterial ferritin Bfr or conventional ferritin FTn or a mutant thereof, preferably, the ferritin is ferritin derived from archaea or bacteria, more preferably, the ferritin is ferritin derived from archaea, more preferably, the ferritin is ferritin derived from Sulfolobales or Pyrococcus, more preferably, the ferritin is encoded by a sequence selected from any one of SEQ ID NOs: 3-7 and 9-11, more preferably, the ferritin is encoded by a sequence selected from any one of SEQ ID NOs: 3-6.

6. The isolated nanozyme with SOD activity as described in any one of claims 1 to 5, which is a nanozyme with SOD activity that does not contain a ferritin shell.

7. The isolated nanozyme with SOD activity as described in any one of claims 1 to 6, wherein the isolated nanozyme with SOD activity has a molar ratio of phosphorus to iron of 0.05-2.5, preferably, a molar ratio of phosphorus to iron of 0.1-2.0, more preferably, a molar ratio of phosphorus to iron of 0.2-1.5, more preferably, a molar ratio of phosphorus to iron of 0.3-1.2, more preferably, a molar ratio of phosphorus to iron of 0.5-1.

0.

8. The isolated nanozyme with SOD activity as described in any one of claims 1 to 7, wherein the DLS particle size range of the isolated nanozyme with SOD activity is 0.1-100 nm, preferably, 0.2-50 nm, more preferably, 0.3-25 nm, more preferably, 0.4-20 nm, more preferably, 0.5-15 nm, more preferably, 0.6-10 nm.

9. A method for preparing the isolated nanozyme having SOD activity according to any one of claims 1 to 8, comprising the following steps: Method 1: 1) Optionally, culture archaea or bacteria and collect bacterial cells; 2) disrupt archaea or bacteria in a buffer solution of pH 7-9.0, and centrifuge to remove insoluble matter and cell debris to obtain a supernatant; 3) for bacterial Bfr purification, incubate the supernatant at 60-68°C for 5-20 minutes, cool down, centrifuge at about 10000-18000 x g for about 5-50 minutes to remove insoluble matter, purify the obtained supernatant with an anion purification column and molecular sieve, and collect the bacterial Bfr fraction; for archaeal FTn purification, incubate the supernatant at 70-85°C for 5-30 minutes, cool down, purify the supernatant with anion purification column and molecular sieve, and collect the FTn fraction; and 4) optionally, removing the ferritin shell to obtain a nanozyme with SOD activity without the ferritin shell; Method 2: a) quickly adding an aqueous solution of iron salt with a concentration of 0.01-0.1 M to an aqueous solution of phosphate with a concentration in the range of 0.001-0.2 M under rapid mixing; b) slowly adding a NaOH or KOH solution to adjust the pH to about 6.2-7.8 and reacting for 10 min-2 hr; and c) optionally, washing the precipitate with water by centrifugation and freeze-drying for later use; Method 3: i) obtaining a buffer solution of bacterial ferritin Bfr or ferritin FTn with a concentration of 0.1–10 mg / mL; ii) adding a phosphate aqueous solution with a concentration of 2-80 mM to the solution in step i) and mixing; iii) slowly adding an aqueous solution of iron salt with a concentration of 2-80 mM to the solution of step ii); and iv) optionally, centrifuging to remove the precipitate and desalting the supernatant; or Method 4: I) optionally, obtaining recombinant cells heterologously expressing bacterial ferritin Bfr or ferritin FTn; II) culturing the recombinant cells heterologously expressing bacterial ferritin Bfr or ferritin FTn in a liquid culture medium to the logarithmic phase; III) adding a ferrous salt aqueous solution with a concentration of 0.5-10mM under the condition of inducing the expression of the bacterial ferritin Bfr or ferritin FTn and continuing to culture for 1-24hr; IV) purifying the bacterial ferritin Bfr or ferritin FTn from the induced expression recombinant cells; and V) optionally, removing the protein shell of the bacterial ferritin Bfr or ferritin FTn and obtaining a nanozyme that does not contain a protein shell.

10. A composition comprising the isolated nanozyme with SOD activity according to any one of claims 1 to 8.

11. The composition according to claim 10, which is a pharmaceutical composition, a cosmetic or a toothpaste.

12. Use of the isolated nanozyme with SOD activity according to any one of claims 1 to 8 in the preparation of an agent for scavenging superoxide free radicals.

13. Use of the isolated nanozyme having SOD activity according to any one of claims 1 to 8 in the preparation of a medicament for preventing or treating conditions and / or diseases caused by superoxide radicals in a subject.

14. Use of the isolated nanozyme with SOD activity as described in any one of claims 1 to 8 as a natural antioxidant.

15. Use of the isolated nanozyme with SOD activity according to any one of claims 1 to 8 in enhancing the stress resistance of microorganisms or plants.

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