WO2015027595A1

WO2015027595A1 - Magnetospirillum magneticum, preparation method therefor, and use thereof

Info

Publication number: WO2015027595A1
Application number: PCT/CN2013/088421
Authority: WO
Inventors: 刘朋明; 高梅影; 陈亚军
Original assignee: 中国科学院武汉病毒研究所; 武汉羽晖特纳生物科技有限公司
Priority date: 2013-08-27
Filing date: 2013-12-03
Publication date: 2015-03-05
Also published as: CN103409354B; CN103409354A

Abstract

Disclosed are a magnetospirillum magneticum, a preparation method therefor, and a use thereof. In the preset invention, a magnetospirillum magneticum Magnetospirillum sp. ME-1, CCTCC No: M2013260 which can produce superparamagnetic magnetosomes is separated fresh water. The strain is a magnetospirillum magneticum with a size of 2.62~4.83×0.44~0.62μm, and comprises a magnetosome chain consisting of 10 to 31 magnetosome particles. A component of each magnetosome is ferroferric oxide, is a cuboctahedron or an octahedron in shape, and has a size of 26±5nm which is in a superparamagnetic range. The magnetospirillum magneticum strain has a specific magnetosome gene island sequence and can produce magnetosomes with a yield of 177mg/L by directly using air fermental cultivation. The strain has excellent fermentability, and produces magnetosome particles which are superparamagnetic, and has a great industrialization popularization prospect.

Description

TECHNICAL FIELD The present invention relates to the field of nano biotechnology, and in particular to a magnetospirilling strain ME-1 capable of producing a superparamagnetic nano magnetosome, and also relates to a The apparatus for collecting the magnetobacteria, the fermenting method of the magnetospirillum, and the application of the magnetospirillum to the preparation of the superparamagnetic magnetosome. BACKGROUND OF THE INVENTION Magnetotactic bacteria are a kind of special bacteria capable of forming nano-magnetic bodies in cells, and the magnetosomes formed thereof have external magnetic field controllability, crystal form stability, nanometer size, excellent biosafety, and outer biofilm. It is not easy to agglomerate, and it shows great advantages compared with other materials in immunoassay, drug delivery, multifunctional molecular carrier, tumor magnetic hyperthermia, magnetic resonance imaging, etc., especially in targeted drug therapy. Application prospects. However, on the one hand, the existing magnetotactic bacteria strains are difficult to ferment and culture, and it is difficult to obtain sufficient magnetosomes for commercial application by mass culture of magnetotactic bacteria. Therefore, the application of magnetosomes is still only in the proof of concept stage. It has been a difficult problem in the field to find a magnetotactic bacterial strain with excellent production traits. Chinese patents ZL02115867.3, ZL02115868.1 and CN1952112A respectively propose "a sludge magnetotactic bacteria and a method for separating and purifying and preparing magnetosomes", "an iron ore magnetotactic bacteria and their separation and purification and preparation of magnetic small The method of "body" and "a facultative anaerobic magnetotactic bacteria and their separation, pure culture method and purification and purification method of magnetic bodies", although the magnetosomes can be obtained, no in-depth research and large-scale production have been found. And application reports. On the other hand, the magnetosomes produced by the current internationally isolated and purified magnetotactic bacteria are single-domain size magnetosomes, which have remanence and coercivity when used as a targeted drug carrier, and are easy to agglomerate and thus easily lead to Form a thrombus. The US special strain lj: US4385119 isolated strain Afoge«tosp r〃Mff2 magnetotacticum MS-1 produced a magnetoid body size of 50 nm and the culture conditions are not strong enough to achieve fermentative production of magnetosomes. The magnetosome provided by Magentospiri m gryphiswaldense MSR-1 is about 43 to 45 nanometers in the US patent US20020012698A1, and the magnetosome provided by Magentospirillum magneticum AMB-1 in Japanese patent JP7241192A is about 50 nanometers. The magnetosomes produced by the three strains of the most deeply studied magnetotactic bacteria in the world are single magnetic domain particles. The production of magnetosomes with smaller particle diameter, less thrombus formation and better dispersion performance will have greater application value than traditional magnetosomes. However, the production of superparamagnetic magnetosomes by magnetotactic bacteria has not been reported so far. . SUMMARY OF THE INVENTION An object of the present invention is to provide a magnetospirillum which can produce superparamagnetic magnetosomes and is easy to be cultured on a large scale. ME-1, this strain produces superparamagnetic magnetosomes with a size of 26±5nm, which is about 1/2 of the size of the magnetosome produced by all the pure culture strains of magnetotactic bacteria reported in the past. It has great potential in the medical field. Application and development potential. The bacteria was sent to the China Center for Type Culture Collection on June 14, 2013. The classification was named: MagnetospirU m sp. ME-1; accession number: CCTCC NO: M2013260; Address: Wuhan University, Wuhan, China. Another object of the present invention is to provide a fermentation method of magnetospirillum ME-1, which is simple and easy to carry out, and has excellent fermentation performance, and solves a large bottleneck of industrial production of magnetic bodies. The prospect of industrialization promotion. Still another object of the present invention is to provide a magnetotactic bacteria collection device which is simple and efficient in design and easy to manufacture by hand. For the collection of magnetotactic bacteria, it can directly enrich and collect magnetotactic bacteria in natural water bodies, eliminating the steps of collecting muddy water samples back to the laboratory for enrichment, avoiding the diversity of magnetotactic bacteria during laboratory enrichment. The loss of sex, in turn, can collect more naturally occurring magnetotactic bacteria in natural waters, effectively improving the success rate of separation of pure cultures by magnetotactic bacteria.

A final object of the present invention is to provide an application of a magnetobacteria in the preparation of a superparamagnetic magnetosome having a good dispersibility of the outer biofilm and a large amount of the envelope. The bioactive group is used for covalently linking with other molecules, and the drug-loaded magnetosome can release the drug by degrading the magnetic extracorporeal membrane in vivo, has good biocompatibility and safety, and has a narrow particle size distribution. A series of advantages such as stable crystal form, single crystal component and no impurities. Compared with the previously reported single magnetic domain magnetosome particles produced by the pure culture strain of magnetotactic bacteria, the size is only about 1/2 of the size of the single magnetic domain magnetosome particles and has superparamagnetism. It is not easy to agglomerate, has better dispersibility, and is less prone to thrombosis. In order to achieve the above object, the present invention adopts the following technical measures: A magnetospirillum ME-1, obtained according to the following preparation steps:

1. Using a self-made magnetotactic bacteria collection device (Fig. 1), directly enrich and collect magnetotactic bacteria samples from natural waters.

2. Further purification of the obtained sample using the capillary Race-track (RT) method with reference to FEMS Microbiology Letters, 1987, 45: 31-35).

3. Purification of the obtained magnetotactic bacteria inoculated with oxygen-sulfur gradient medium (method refer to Systematic and Applied Microbiology, 1999, 22: 466-471, and the medium formulation is shown in Table 1), and cultured at 30 ° C for more than 7 days.

4. A magnetotactic bacterial disc-shaped growth band appeared in the semi-solid medium, and the magnetotactic bacterial culture was taken out to inoculate the magnetotactic bacterial growth medium by using Dilution to Extinction Methods (see Table 4). 5. The obtained pure seed culture was separated and purified by three extreme density gradient dilution methods to obtain a pure culture strain of magnetotactic bacteria. The magnetic properties of the strains were observed by optical phase contrast microscopy. The pure growth strain Magnetospirillum sp. ME-1 with the fastest growth and the best magnetic properties was selected. The magnetobacteria strain Magnetospiri Uum sp. ME-1 of the present invention has been preserved in the China Center for Type Culture Collection, Address: Wuhan University, Wuhan, China, Date of Deposit: June 14, 2013, Accession No.: CCTCC NO: M 2013260, the classification is named: Magnetospirillum M/g «etos^r〃M»j sp. ME-1. The method is characterized in that the magnetobacteria are microaerobic bacteria, the shape of the cells is spiral, and the flagella are both ends, and the size of the cells is 2.62~4.83Χ0.44~0.62μπι, and each cell produces a 10~31 A magnetosome chain composed of a cubic octahedron or a cubic magnet body having a size of 26 ± 5 nm. The main composition of magnetosome crystals is iron and oxygen, and the particles are in the range of superparamagnetic domain size. The physiological and biochemical characteristics of the strain are shown in Table 5. Spirulina

The gene island responsible for the synthesis of magnetosomes in the sp. ME-1 genome was analyzed. Single colony inoculation of growth medium, purification of genomic DNA (method refer to Journal of Molecular Biology, 1961, 3: 208-218), genome sequencing to obtain 16SrDNA sequence (this sequence is shown in SEQ No. 2 of the Sequence Listing), for phylogeny Tree analysis found that it belongs to the genus Streptomyces. The magnetosome gene island sequence was obtained (this sequence is shown in SEQ No. 3 and SEQ No. 1 of the sequence listing), and compared with several other strains of magnetospirillum, the strain was found to have a sequence on the magnetosome gene island. Specificity. A device for collecting magnetotactic bacteria, constructed as follows: a cylindrical plastic collection box (1) with one end open; filter paper (2); a metal tripod (3); a permanent strong magnet (4); and a fishing line (5). The connection relationship is: filter paper (2) through the metal tripod (3) to the sealed end of the cylindrical plastic collection box (1) open end, the S pole of the permanent strong magnet (4) and the closed end of the cylindrical plastic box (1) Connected, the fishing line (5) is connected to the N pole of the permanent magnet (4).

The filter paper is a fast qualitative filter paper, the filter paper has a pore size of 80~120 micrometers; the permanent strong magnet is a button type; the magnetotactic bacteria collection device uses the following steps:

1 Inverted collection device, cylindrical plastic collection box (1) filled with in-situ water filtered and sterilized;

2 Use a metal tripod (3) to seal the filter paper (2), install the magnet (4) and the fishing line (5);

3 into the natural water environment to make the device stand at the mud-water interface and the filter paper surface of the collection device is located 1~2cm above the mud-water interface, and is allowed to stand for 12 hours; 4 After the device is left for 12 hours, remove it, remove the tripod, carefully open the filter paper, take up the collected liquid in the collection box and observe the optical microscope within 2 hours, the preparation of the electron microscope copper mesh and the separation and culture of the magnetotactic bacteria. . A fermenting method for the bacterium Mycobacterium ME-1, the breeding conditions are:

The 10 L fermentor was filled with 6 L of growth medium, inoculated with 10% inoculum, culture conditions were 30 ° C, air was passed at 20 L/h, stirring speed was coupled with 2% dissolved oxygen, and the feed was coupled with pH 6.86. Magnetospirillum

ME-1 grew to a cell density of OD ₅₆₅ 2.5 in about 40 hours under such culture conditions, and a magnetic body yield of 55 mg/L was obtained. The growth medium formula is: vitamin mixture lml / l, mineral mixture 2 ml / 1, potassium phosphate buffer (0.1M, pH7.0) 10ml / l, sodium succinate 0.05% (m / V) , sodium acetate 0.05% (m/V), yeast extract 0.025% (m/V), ammonium chloride 0.05% (m/V), magnesium sulfate 0.01% (m/V), ferric citrate, vitamins The mixed solution and the mineral mixture solution are separately sterilized by the medium, and the filter sterilization liquid is separately added. The formula of the feed liquid is: a vitamin mixture of 10 ml / l, a mineral mixture of 20 ml / 1, a potassium phosphate buffer (0.1 M, pH 7.0) lOOml / 1, sodium succinate 0. 5% (m / V), sodium acetate 0. 5% (m/V), yeast extract 0.25% (m/V), ammonium chloride 0. 5% (m/V), ferric citrate lmM, wherein the vitamin mixture The mineral mixture solution is sterilized by the medium and the filter solution is separately added. The formulation and configuration method of the vitamin mixture are shown in Table 2. The mineral mixture formulation and configuration method are shown in Table 3. The preferred medium and feeding formula are as follows: The growth medium formula is: vitamin mixture 3 ml/l, mineral mixture 2 ml/l, adipic acid 0.2% m/V, ammonium nitrate 0.05% m/V, yeast extraction 0.05% m/V, peptone 0.25% m/V, 150μM ferric citrate, wherein the vitamin mixture and mineral mixture need to be separately sterilized by filtration to remove the bacterial solution; the formula of the feed solution is: vitamin mixture 30ml/l, mineral mixture 20 ml/1, adipic acid 2.0% m/V, ammonium nitrate 0.5% m/V, yeast extract 0.5% m/V, peptone 2.5% m/V, ferric citrate 0.05 % m/V, wherein the vitamin mixture and the mineral mixture are separately added to the filter sterilization solution after the medium is sterilized. The formulation and configuration method of the vitamin mixture are shown in Table 2. The mineral mixture formulation and configuration method are shown in Table 3. An application of a magnetostrictive 蠊*3⁄4 in the preparation of a super-magnetic magnetic body, the steps of which are: 1. Fermentation culture, culture of S. oxysporum strain according to the above fermentation culture method

ME-1 was grown to a cell density of OD ₅₆₅ 2.5 for about 40 hours;

2. Collecting by centrifugation, collecting the cells obtained by fermentation culture by centrifugation;

3. Wash, wash twice with lOmM PBS (pH 7.4), and resuspend in PBS buffer;

4. Broken cells, crushed at pressure (1500 bar) three times;

5. Collect, the broken bacterial suspension adsorbs the magnetic body with a magnet, discard the supernatant, resuspend in PBS buffer, and shake with 50W ultrasonic wave (working for 5 seconds, intermittent 5 seconds, a total of 90 times). Dissociation of cell debris The magnetic body is wrapped, then adsorbed, resuspended, and repeated 15 times or more;

6. Quantitatively, the collected magnetosomes are weighed with a filter paper and then weighed. The magnetosome produced by the magnetospirillum ME-1 provided by the present invention has a magnetosome size of 26 ± 5 nm. Magnetic domain analysis (method reference Geochemistry Geophysics Geosystems, 2009, 10: 1-19; Journal of Geophysical Research-Solid Earth, 2006, 111: B 12S 12 and Journ d Of Geophysical Research, 1975, 80: 4049-4058) In the magnetospirillic cells, since the magnetosomes are close to each other in the chain and exhibit a single magnetic domain characteristic, the magnetosomes are separated from each other to exhibit superparamagnetic characteristics. Compared with the prior art, the present invention has the following advantages and effects: The present invention provides a magnetic parasitic bacterium capable of producing a superparamagnetic magnetic body, and the superparamagnetic magnetic body produced has a size of 26±5 nm. Compared with the synthetic superparamagnetic nanomaterials, the biofilm has good dispersibility, and the envelope can provide a large number of bioactive groups for covalent attachment with other molecules, and the drug-loaded magnetosomes can degrade magnetically in vivo. The small extracorporeal membrane can achieve a series of advantages such as drug release, good biocompatibility and safety, narrow particle size distribution, stable crystal form, and single crystal component without impurities. Compared with the single magnetic domain particle magnetosome produced by the magnetotactic bacteria reported in the past, the size is only about 1/2 of the size of the magnetic body previously reported and has superparamagnetism, so it is not easy to agglomerate and disperse in application. Good, less likely to form a blood clot. It has great application and development potential in the medical field. The magnetic spirulina provided by the invention can use the common fermenter to produce magnetic bodies by air fermentation, and has excellent fermentation performance, and has great advantages and industrialization promotion prospects in the industrial production of magnetic bodies. The invention provides a magnetotactic bacteria collecting device which is simple and efficient in design and easy to manufacture by hand. The device is used for the collection of magnetotactic bacteria, which eliminates the step of collecting the muddy water samples back to the laboratory for enrichment, avoiding the trend in the laboratory enrichment process. The loss of the diversity of the magnetic bacteria can collect more natural magnetotactic bacteria in the natural water body, thereby effectively improving the success rate of the separation culture of the magnetotactic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of a magnetotactic bacteria collection device. Among them: 1 is a cylindrical plastic collection box with one end open, 2 is filter paper, 3 is a metal tripod, 4 is a button-shaped permanent strong magnet, and 5 is a fishing line. In Fig. 2, A is a spiral microscopic morphology of Magnetospiri Uum sp. ME-1 and its magnetosome electron microscopic morphology. In Fig. 2, B is the energy spectrum of the composition of the magnetosome, and it can be seen that the energy spectrum of the magnetosome is larger than the energy spectrum of the blank region of the cell, and the probe has a diameter of 2-10 nm. In Fig. 3, A is a statistical analysis of the width of the magnetosome, and its width is 23±5 nm _{; in} Fig. 3, B is a statistical analysis of the length of the magnetosome, and its length is 26±5 nm; the statistical quantity is 420. In Fig. 4, A is a morphological view of a purified magnetosome. The purified magnetosomes have a uniform size and an average size of 26±5 nm. A1 is an enlarged magnetosome, and its shape is a cubic octahedron, and the arrow shows a biofilm. In Fig. 4, B is a schematic diagram of magnetic domain distribution analysis of magnetic bodies. The analysis of the magnetic domain characteristics of the magnetosome shows that the magnetosome exhibits a single magnetic domain characteristic in the case of interaction between the magnetosomes, and the superparamagnetic property appears in the absence of interaction after separation of the magnetosomes. Specifically, the following is a detailed description of the obtaining, characterization, and fermentation culture and preparation method of the magnetic body of the present invention. The reagents used in the examples of the present invention can be purchased from a conventional biochemical reagent store or a pharmaceutical business enterprise unless otherwise specified. Example 1: A magnetospirillum ME-1 was screened by the following procedure: 1) Collection and capillary separation of magnetotactic bacteria samples:

(1) Magnetotactic bacteria collection: Inverted collection device, cylindrical plastic collection box 1 filled with filtered in-situ water (in situ The water is collected from the water sample at the interface of the mud and water, filtered and sterilized by 0.22μπ filter membrane. The filter paper 2 is sealed with a metal tripod 3, and the magnet 4 and the fishing line 5 are placed in the natural water environment to make the device stand in the muddy water. At the interface, the filter paper surface of the collection device is located at 1~2cm above the mud-water interface. After 12 hours of storage, remove it, remove the tripod, carefully open the filter paper, and take the collected liquid from the collection box and observe it under optical microscope within 2 hours. The fabrication of an electron microscope copper mesh and the separation and culture of magnetotactic bacteria.

(2) Capillary separation of magnetotactic bacteria: Samples collected by the collection device were observed by phase contrast microscopy, and samples of more magnetotactic bacteria were observed using capillary Race-track (RT) method. FEMS Microbiology Letters, 1987, 45: 31-35) Further purification was carried out to obtain a magnetotactic bacterial culture seed solution. The magnetotactic bacteria collecting device is: a cylindrical plastic collecting box 1 with one end open; a filter paper 2; a metal tripod 3; a permanent strong magnet 4; and a fishing line 5. The connection relationship is as follows: The filter paper 2 is connected to the open end of the cylindrical plastic collecting box 1 through the metal tripod 3, and the S pole of the permanent strong magnet 4 is connected with the closed end of the cylindrical plastic box 1, the fishing line 5 and the permanent strong magnet 4 The N pole connection is shown in Figure 1. The filter paper is a fast qualitative filter paper, the filter paper has a pore size of 80~120 microns; the permanent strong magnet is a button type;

2) Preliminary culture of magnetotactic bacteria The magnetotactic bacterial seed solution was inoculated with an oxygen-sulfur gradient medium, and cultured at 30 ° C for 7 days or more until a magnetotactic bacterial disc-shaped growth band was obtained in the semi-solid medium. The culture was taken out, and the culture having the growth of the magnetotactic bacteria was confirmed by a phase contrast microscope hanging drop method as a seed liquid for pure culture separation of the magnetotactic bacteria. The formulation of the oxygen-sulfur gradient medium is shown in Table 1.

Table 1 Oxygen-sulfur gradient medium formula

Upper oxygen layer culture 3⁄4 formula

Ingredients containing J unit in situ water 200 ml vitamin mixture 1 ml mineral mixture (excluding ' 2 ml sodium succinate 0.05 g yeast extract 0.05 g

MgS0 ₄ 0.05 g Potassium phosphate buffer (pH 7.0) 0.5 mM Resazurin 2 mg Agar 2 g Distilled water 800 ml The in-situ water is taken from the mud-water interface of the sample collection water and filtered by 0.22μπ filter. The medium is adjusted to ρΗ7 .0, sterilized, supplemented with 1 ml of sterile 0.01 M ferric citrate (ΙΟμΜ) and neutralized and filtered the sterilized cysteine hydrochloride (1% mother liquor 10 ml) (0.01%). The anaerobic tube cover is loosened. 10 ml of the upper medium was added to the bottom layer (sulfur layer) and allowed to stand for at least 24 hours to form a gradient and inoculated.

Lower sulfur layer medium formula

Na2S 4 mM agar 1.5 g Distilled water 100 ml Adjust pH 7.4, sterilize each anaerobic tube into lml at the bottom, and then add the upper layer (oxygen layer) after solidification

The formula and configuration method of the vitamin mixture are shown in Table 2

Table 2 vitamin mixture formula

Ingredients: Biotin 2.000 mg Folic acid 2.000 mg Vitamin B6 (Pyridoxine-HCl) 10.000 mg Vitamin Bl (Thiamine-HCl x 2 H ₂ 0) 5.000 mg Vitamin B2 (Riboflavin) 5.000 mg Vitamin B3 (Nicotinic acid) 5.000 mg D-Ca-pantothenate 5.000 mg Vitamin B ₁₂ (Vitamin B ₁₂ ) 0.100 mg p-Aminobenzoic 5.000 mg Acid)

Lipoic acid 5.000 mg

MilliQ water 10 00.000 ml was dissolved in sequence, filtered at 0.22 μm and stored at 4 ° C until use. The formulation of the mineral mixture is shown in Table 3.

Table 3 Mineral mixture formula

Ingredients: Nitrilotriacetic acid 1.500 g Magnesium sulfate heptahydrate (MgS0 ₄ '7H ₂ 0) 3.000 g Manganese sulfate dihydrate (MnSCV2H ₂ 0) 0.500 g Sodium chloride (NaCl) 1.000 g Seven Ferrous sulfate (FeS0 ₄ '7H ₂ 0) 0.100 g Cobalt sulfate heptahydrate (CoSCV7H ₂ 0) 0.180 g Calcium chloride dihydrate (CaCl ₂ '2H ₂ 0) 0.100 g Zinc sulfate heptahydrate (ZnSCV7H ₂ 0) 0.180 g Copper sulfate pentahydrate (CuS0 ₄ '5H ₂ 0) 0.010 g Potassium aluminum sulfate dodecahydrate (KA1(S0 ₄ ) ₂ -12

0.020 g 3⁄40)

Boric acid (H ₃ B0 ₃ ) 0.010 g Sodium molybdate dihydrate (Na ₂ Mo0 ₄ '2H ₂ 0) 0.010 g Nickel hexahydrate (Ν 3⁄4·6Η ₂ 0) 0.025 g Sodium selenate (Na ₂ Se0) ₃ '5H ₂ 0) 0.300 mg

MilliQ water 10 00.000 ml First dissolve nitrous triacetate with KOH adjusted to pH 6.5, then add each mineral, and finally KOH ρ7.0, 0.22μπι filter and store at 4 °C.

3) Magnetospirillum sp. ME-1 strain Single colony obtained The magnetomic bacteria primary culture was inoculated with the magnetotactic bacterial growth medium in the anaerobic tube. The culture was carried out at 30 ° C for more than 7 days until a magnetotactic bacterial disc-shaped growth band was obtained in the semi-solid medium. The culture was taken out and observed by phase contrast microscopy to confirm the presence of magnetotactic bacteria. For growth, the culture was separated as a seed solution of pure culture by limiting gradient dilution three times (method reference to The lSME Journal, 2012, 6: 440-450). A fast-growing, magnetically good magnetotactic bacterial strain ME-1 was obtained, which was sent to the China Center for Type Culture Collection for preservation on June 14, 2013. The classification was named: MagnetospirU m sp. ME- 1 , Deposit No.: CCTCC NO: M 2013260, Address: Wuhan University, Wuhan, China. The structure of the magnetotactic bacteria growth medium is shown in Table 4. Table 4 Magnetotactic bacteria growth medium formula ingredients will be a bone unit vitamin mixture 1 ml mineral mixture 2 ml potassium phosphate buffer (pH 7.0) 1 mM Sodium succinate 0.1 g sodium acetate 0.1 g yeast extract 0.05 g

NH ₄ C1 0.1 g

MgS0 ₄ 0.1 g ferric citrate 20 μΜ agar 2 g distilled water 1000 ml

4) Morphological and physiological and biochemical characteristics of Magnetospirillum sp. ME-1 strains Morphological observation of the obtained strain ME-1 by optical microscopy and electron microscopy revealed that it was a spirochete with flagella at both ends and 10 to 31 magnetic bodies in the body. The small body consists of a magnetosome chain with a cell size of 2.62~4.83Χ0.44~0.62μπι, with flagella at both ends. X-ray energy spectroscopy (XEDS) analysis revealed that the magnetosome crystals were composed of Fe and 0 (Fig. 2). Refer to (Jnternationa! Journal of Systematic Bacteriology, 1981, 31: 452-455) for physiological and biochemical tests on ME-1, and obtain physiological and biochemical properties as shown in Table 5.

Table 5 Morphological, physiological and biochemical characteristics of Magnetospirillum sp. ME-1 strain

Strain ME-1

The size of the bacteria is 2.62~4.83Χ0.44~0.62μπι. The magnetic body size is 26nm. Catalase Activity - Oxidase Activity + Phosphatase Activity - Sulfate Activity - Urease Activity + Casein and Starch Hydrolytic Activity - Hydrolysis Activity of Hippuric Acid and Heptaline + Reduction of Nitrate to Nitrite - Nitrate Cross-Nitrous Acid Salt reduction + ammonia production - gelatin liquefaction activity - hydrazine test - hydrogen sulphide production -

1% glycine growth - l% NaCl growth - soluble fluorescein production - late culture spheroid + poly beta hydroxybutyrate (ΡΗΒ) + Example 2:

Phylogeny and gene island analysis of Magnetospirillum sp. ME-1 strain

1) Genomic extraction Genomic DNA of strain ME-1 was extracted by glass rod stirring method (Method reference of Molecular Biology, 1961, 3: 208-218) „ Roche GS FLX shotgun database, Roche GS FLX Titanium System 454 sequencer sequencing, The sequence is spliced to produce 79 _CO ntigs.

2) The measured strain 16SrDNA sequence (this sequence is shown in SEQ ID No. 2 of the Sequence Listing) was subjected to phylogenetic analysis, and it was found that the strain belongs to the genus Magentospirillum, and the sequence of the strain 16SrDNA is different! J and Magentospirillum magneticum AMB-1, Magentospirillum magnetotactic mMS-l Magentospirillum gryphiswaldenseMSK- 1 The 16SrDNA sequence has 99.4%, 98.1%, and 96.3% sequence similarity, and is proudly named MagentospiriUum sp. ME-1.

3) The genome of MagentospiriUum magneticum AMB-1 (GenBank accession NO: AP007255) was compared and analyzed by Mauve software. According to the results of the comparison analysis, the composition of Magentospirillum sp. ME-magnetosome gene island was obtained. Contigs. The GL GenomewalkerTM Universal Kit kit was used to fill the gaps between the magnetosome gene island congtigs, and the complete ME-1 magnetosome gene island sequence was obtained.

(This sequence is shown in the Sequence Listing SEQ ID N0.3, Sequence Listing SEQ ID NO. 1).

4) Analysis of magnetosome gene islands

The BLAST alignment analysis revealed that the Magentospirillum sp. ME-1 magnetosome gene island consisted of mamGFDC, mms6, mamAB, and mamXY cistrons. Among them, mamGFDC cistron contains four magnetosome-related genes such as mamG, mamF, mamD and mamC; mms6 cistron contains magnetosome-related gene mms6; mamAB cistron contains mamH, maml, mamE, mamJ, mamK, mamL 18 magnetosome-related genes such as mamM, mamN, mamO, mamP, mamA, mamQ, mamR, mamB, mamS, mamT, mamU, mamV; mamGFDC mms6, mamAB sequence as shown in SEQ ID No. 3 of the sequence listing, mamXY (This sequence is shown in SEQ ID No. 1 of the Sequence Listing) contains 6 magnetosome-related genes such as mms5, mamY, mamX-like, mamZ, mamH-like, and FtsZ-like, and is related to other magnetosome-related genes in the genome. The distance between them is far. Example 3:

The growth of Magnetospirillum sp. ME-1 strains was carried out in a 10 L fermentor with the fermentation of the Magnetospiri Uwn sp. ME-1: The specific conditions were: 10 L of fermenter was added with growth medium 6 L, inoculated with 10% inoculum, cultured The condition was 30 ° C, air was passed at 20 L/h, the stirring speed was coupled with 2% dissolved oxygen, and the feed was coupled with a pH of 6.86. Magnetospirillum Magnetospirillmn sp. ME-1 grew to OD ₅₆₅ 2.5 in this culture condition for about 40 hours. The growth medium formula is: Vitamin mixture (see Table 2 for formula) lml/1, mineral mixture (see Table 3 for formula) 2 ml/1, potassium phosphate buffer (0.1M, pH7.0) 10ml /l , Sodium succinate 0.05% (m/V), Sodium acetate 0.05% (m/V), Yeast extract 0.025% (m/V), Ammonium chloride 0.05% (m/V), Magnesium sulfate 0.01% (m/V), 100 μM of ferric citrate, in which the vitamin mixture and the mineral mixture are sterilized by the medium, and the filter solution is separately added. The feed liquid formula is: vitamin mixture (see Table 2 for formula) 10ml/l, mineral mixture (see Table 3 for formula) 20 ml/1, potassium phosphate buffer (0.1M, pH 7.0) lOOml/1, sodium succinate 0. 5% (m/V), sodium acetate 0. 5% (m/V), yeast extract 0.25 % (m/V), ammonium chloride 0. 5% (m/V), iron citrate lmM, wherein the vitamin mixture and the mineral mixture need to be sterilized by the medium, and the filter solution is separately added. Example 4:

Hairospirillum sp. ME-1 strain hair growth: Fermentation culture experiment of Magnetospiri Uwn sp. ME-1 in a 10L fermenter: The specific conditions are: 10L fermenter is equipped with 6L of growth medium, 10% inoculation amount is inoculated, The culture condition was 30 ° C, air was passed at 20 L/h, the stirring speed was coupled with 2% dissolved oxygen, and the feed was coupled with a pH of 6.86. The magnetospirilling fungus Magnetospirillmn sp. ME-1 was subjected to 40 hours under such culture conditions. It grows to the left and right to OD ₅₆₅ 5.7. The magnetotactic bacteria are: magnetobacteria Afog «etos^r〃M»js;?. ME-1, CCTCC NO: M2013260; the growth medium formula is: vitamin mixture 3ml / l (formulation See Table 2), mineral mixture 2 ml/1 (formulation shown in Table 3), adipic acid 0.2% m/V, ammonium nitrate 0.05% m/V, yeast extract 0.05% m/V, peptone 0.25% m /V, 150μΜ of ferric citrate, wherein the vitamin mixture and the mineral mixture need to be separately sterilized by adding the filter sterilization solution; the formula of the feed solution is: vitamin mixture 30ml/l (see Table 2 for formula) ), mineral mixture 20 ml/1 (formulation shown in Table 3), adipic acid 2.0% m/V, ammonium nitrate 0.5% m/V, yeast extract 0.5% m/V, peptone 2.5% m/V, Ferric citrate 0.05% m/V, wherein the vitamin mixture and the mineral mixture are separately added to the filter sterilization solution after the medium is sterilized. Example 5: The use of a magnetospirillum in the preparation of a superparamagnetic magnetosome, the steps of which are:

1) Fermentation culture, according to the fermentation culture method described in Example 3, the culture of the diatom bacterium strain M3⁄4 « _e to _S ^W / / _M sp. ME-140 hours to the bacterial cell density OD ₅₆₅ 2.5 to stop the fermentation;

2) collecting by centrifugation, collecting the cells obtained by fermentation culture by centrifugation;

3) Wash, wash twice with lOmM PBS (pH 7.4), and resuspend in PBS buffer;

4) Broken cells, crushed at pressure (1500 bar) three times;

5) Collect, the broken bacterial suspension adsorbs the magnetic body with a magnet, discards the supernatant, and resuspend in PBS buffer, 50W ultrasonic vibration (working for 5 seconds, intermittent 5 seconds, a total of 90 times) Dissociation of cell debris The magnetic body is wrapped and then adsorbed, Resuspend, repeat 15 times or more;

6) Quantitatively, the collected magnetosomes are weighed with a filter paper and weighed; Magnetospirillum sp. ME-1 is fermented for about 40 hours to obtain a yield of 55 mg/L. The fermentation broth obtained in the medium and the culture conditions of Example 4 had a magnetic body yield of 177 mg/L.

The magnetosomes obtained by transmission electron microscopy were cubic octahedrons or cubes, and the outer biofilm (Fig. 4) was uniform in particle size. The measurement showed that the width was 23±5 nm and its length was 26±5 nm (Fig. 3 ). Magnetic domain analysis (method reference Geochemistry Geophysics Geosystems, 2009, 10: 1-19; Journal of Geophysical Research-Solid Earth, 2006, 111: B 12S 12 and Journ d Of Geophysical Research, 1975, 80: 4049-4058) Its magnetic domain distribution is in the range of superparamagnetic properties (Fig. 4).

Claims

Claim

A magnetospirillum, characterized by: magnetospirillum MagnetospiriUum sp., ME-1, CCTCC NO: M 2013260

2. The isolated magnetosome gene island of the genus Streptomyces according to claim 1, the sequence of which is represented by SEQ ID N0.1.

3. An isolated magnetosome gene island of the genus Streptomyces according to claim 1, the sequence of which is represented by SEQ ID N0.3.

4. An isolated 16Sr DNA of the genus Streptomyces according to claim 1, the sequence of which is represented by SEQ ID NO.

5. The method of fermenting a magnetospirillum ME-1 according to claim 1, wherein the fermentation culture condition is:

10L fermenter is equipped with 6L of growth medium, and the magnetotactic bacteria are inoculated with 10% inoculum. The culture condition is 30°C, the air is 20L/h, the stirring speed is coupled with 2% dissolved oxygen, and the feeding and pH value is 6.86. The magnetotactic bacteria are: magnetospirillum Afog «etos^r〃M»js;?. ME-1, CCTCC NO: M2013260; the growth medium formula is: vitamin mixture lml/l , mineral mixture 2 ml / l, 0.1 M, pH 7.0 potassium phosphate buffer 10 ml / l, sodium succinate 0.05% m / v, sodium acetate 0.05% m / V, yeast extract 0.025% m / v , ammonium chloride 0.05% m/V, magnesium sulfate 0.01% m/V, iron citrate ΜμΜ, wherein the vitamin mixture and the mineral mixture are sterilized by the medium, and the filter sterilizing solution is separately added; The liquid formulation is: vitamin mixture 10ml / l, mineral mixture 20 ml / l, 0.1M, pH 7.0 potassium citrate buffer 100ml / l, sodium succinate 0. 5% m / v, sodium acetate 0 5% m/V, yeast extract 0.25% m/V, ammonium chloride 0. 5% m/V, ferric citrate lmM, wherein the vitamin mixture and mineral mixture are separately sterilized in the medium Add filter sterilization solution; vitamin Formulation combined solution was: Component Amount Unit biotin 2.000 mg; Folic acid 2.000 mg; Vitamin B6 10.000 mg; Vitamin B 1 5.000 mg; Vitamin B2 5.000 mg; Vitamin B3 5.000 mg; calcium pantothenate 5.000 mg; vitamin B12 0.100 mg; p-aminobenzoic acid 5.000 mg; lipoic acid 5.000 mg; water

The formulation of the mineral mixture is:

Ingredient content unit Nitrogen triacetate 1.500 g; magnesium sulfate heptahydrate 3.000 g; manganese sulfate dihydrate 0.500 g; sodium chloride 1.000 g; ferrous sulfate heptahydrate 0.100 g; cobalt sulfate heptahydrate 0.180 g; calcium chloride dihydrate 0.100 g; zinc sulfate heptahydrate 0.180 g; copper sulfate pentahydrate 0.010 g; potassium aluminum sulfate dodecahydrate 0.020 g;

0.010 dihydrate molybdate 0.010 g; hexahydrate nickel chloride 0.025 pentahydrate sodium selenate 0.300 mg; Water 1000.000 ml

6. The method of fermenting a magnetospirillum ME-1 according to claim 1, wherein the fermentation culture condition is:

10L fermenter is equipped with 6L of growth medium, and the magnetotactic bacteria are inoculated with 10% inoculum. The culture condition is 30°C, the air is 20L/h, the stirring speed is coupled with 2% dissolved oxygen, and the feeding and pH value is 6.86. The magnetotactic bacteria are: magnetobacteria Afog «etos^r〃M»js;?. ME-1, CCTCC NO: M2013260; the growth medium formula is: vitamin mixture 3ml/l , mineral mixture 2 ml/1, adipic acid 0.2% m/V, ammonium nitrate 0.05% m/V, yeast extract 0.05% m/V, peptone 0.25% m/V, ferric citrate 150μΜ, vitamin The mixed solution and the mineral mixture solution are separately sterilized by the culture medium, and the filter solution is separately added; the formula of the feed liquid is: vitamin mixture 30 ml/l, mineral mixture 20 ml/1, adipic acid 2.0% m/V, ammonium nitrate 0.5% m/V, yeast extract 0.5% m/V, peptone 2.5% m/V, ferric citrate 0.05% m/V, in which the vitamin mixture and mineral mixture are eliminated in the medium The filter sterilization solution was added separately after the bacteria. The formula of the vitamin mixture is: ingredient content biotin 2.000 mg; folic acid 2.000 mg; vitamin B6 10.000 mg; vitamin B1 5.000 mg; vitamin B2 5.000 mg; vitamin B3 5.000 mg; calcium pantothenate 5.000 mg; vitamin B 12 0.100 mg; P-aminobenzoic acid 5.000 mg; lipoic acid 5.000 mg; water The formula of the mineral mixture is: The composition contains 』 unit of Nitrogen triacetate 1.500 g _; magnesium sulfate heptahydrate 3.000 g _:

: water manganese sulfate 0.500 g _; sodium chloride 1.000 g _; ferrous sulfate heptahydrate 0.100 g _: cobalt sulfate heptahydrate 0.180 g _;

: water calcium chloride 0.100 g _; zinc sulfate heptahydrate 0.180 g _; copper sulfate pentahydrate 0.010 g _; potassium sulfate aluminum sulfate 0.020 g _;

0.010 g _; sodium molybdate dihydrate 0.010 g _; nickel chloride hexahydrate 0.025 g _; sodium selenate pentahydrate 0.300 mg _; water 1000.000 ml o

7. A device for collecting magnetotactic bacteria, comprising: a cylindrical plastic collecting box (1) having an opening at one end, and a filter paper (2) sealing the open end of the cylindrical plastic collecting box (1) by a metal tripod (3) Connecting, the S pole of the permanent strong magnet (4) is connected to the closed end of the cylindrical plastic case (1), and the fishing line (5) is connected to the N pole of the permanent strong magnet (4); the filter paper is a fast qualitative filter paper, The filter paper has a pore size of 80 to 120 μm; and the permanent strong magnet is a button type.

8. Use of a magnetospirillum of claim 1 for producing a superparamagnetic magnetosome.