Microbacterium SCSIO 12466 and method for producing heme by using microbacterium SCSIO 12466
By using Microbacterium sp. SCSIO 12466 as a new host, the problems of low product purity, high cost and long production cycle in the existing heme preparation methods were solved, and the microbial synthesis of high-purity heme was achieved, avoiding the limitations in the production process of E. coli.
Patent Information
- Application Number
- CN202510231287.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-30
AI Technical Summary
The existing heme preparation methods have problems such as low product purity, high cost and long production cycles. The use of E. coli to produce heme faces limitations such as host containing endotoxins and genetic instability of plasmids.
A method for producing heme using Microbacterium sp. SCSIO 12466, which can grow within suitable temperature, pH and salinity ranges, and serve as a novel host to replace E. coli for microbial synthesis of heme.
The high-purity heme is obtained through the fermentation culture of microbacterium SCSIO 12466, which solves the problems of product purity and cost in traditional methods, and avoids the problems of endotoxins and genetic instability in the production process of E. coli.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a method for producing heme using Microbacterium sp.. Background Art
[0002] Heme is a compound formed by the combination of protoporphyrin IX (PPIX) and ferrous ions, and is widely distributed in various organisms in nature. It plays an important role in maintaining the supply and transportation of iron in the body, and is a key component of related proteins in respiration and the electron transport chain. In addition, heme is also involved in various life activities such as signal transduction, circadian rhythm regulation, and miRNA processing and synthesis. With the continuous in-depth research, people's understanding of the functions of heme will be more comprehensive.
[0003] Benefiting from the unique structure and diverse functions of heme, it has shown extensive value in practical applications: due to the high absorption efficiency of heme iron, heme can be used as an iron supplement and anti-anemia drug in the field of medical health; in the food industry, heme can replace nitrite and synthetic pigments with potential carcinogenic risks, thereby improving the safety of food; in disease diagnosis and treatment, heme has been widely used in iron deficiency treatment, acute intermittent porphyria, malaria parasite lysis, etc., and is also an important raw material for porphyrin drugs in anti-cancer drugs.
[0004] At present, the preparation of heme mainly relies on the glacial acetic acid method, acidic acetone method (including the sodium acetate method, distillation method, tannic acid method, etc.), carboxymethyl cellulose (CMC) method, and protease hydrolysis method. However, these traditional processes have problems such as low product purity, high cost, and long production cycle. Therefore, the biological fermentation method has gradually attracted attention, and certain progress has been made in producing heme using Escherichia coli. However, this method faces limitations such as the host containing endotoxin and plasmid genetic instability. Therefore, it is particularly important to find a new host that can replace Escherichia coli for heme production. Summary of the Invention
[0005] The first object of the present invention is to provide a strain of Microbacterium sp. SCSIO 12466, and its preservation number is: CGMCC No. 32943.
[0006] The Microbacterium sp. SCSIO 12466 is streaked and inoculated on MA medium and cultured at a constant temperature of 25 °C for 3 days. Observe the morphological characteristics of the strain. The colony is yellow, round, and smooth on the surface ( Figure 1 ).
[0007] The suitable growth temperature range of Microbacterium sp. SCSIO 12466 is 10°C to 40°C, the pH range is 5 to 10, and the salinity range is 1% to 4% (mass percentage) NaCl.
[0008] Preferably, the most suitable growth temperature of Microbacterium sp. SCSIO 12466 is 25°C to 30°C, the pH range is 6 to 7, and the salinity range is 3% (mass percentage) NaCl.
[0009] The second object of the present invention is to provide a microbial inoculum containing Microbacterium sp. SCSIO 12466 as an active ingredient.
[0010] The third object of the present invention is to provide a method for producing heme from Microbacterium sp. SCSIO 12466. The heme described in this method is obtained from the fermentation culture of Microbacterium sp. SCSIO 12466.
[0011] The fermentation culture of Microbacterium sp. SCSIO 12466 is obtained by fermenting and culturing with MB liquid medium as the fermentation medium.
[0012] Preferably, the formula of the MB liquid medium is: 5 g peptone, 1 g yeast extract, 0.1 g ferric citrate, 1 L seawater, pH 7.0.
[0013] Preferably, the culture conditions are culturing at 25°C for 36 - 72 hours.
[0014] Preferably, the fermentation culture is bacterial cells.
[0015] The strain SCSIO 12466 of the present invention is Microbacterium sp. SCSIO 12466, and the preservation number is CGMCC No. 32943. Through temperature, salinity, and pH adaptability tests, it shows that this strain can grow in a culture solution with a temperature of 10°C to 40°C, a pH of 5 to 10, and an NaCl mass percentage of 1% to 4%, and is suitable for fermentation under various culture conditions, providing high-quality strain resources for the microbial synthesis of heme.
[0016] Microbacterium sp. SCSIO 12466 was deposited on December 6, 2024, at the China General Microbiological Culture Collection Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing 100101, and the preservation number is CGMCC No. 32943. Description of the Drawings
[0017] Figure 1 This is the colony morphology of the strain SCSIO 12466 of the present invention cultured on MA medium at 25 °C for 3 days.
[0018] Figure 2 This is the detection of the temperature range for the growth of Microbacterium sp. SCSIO 12466.
[0019] Figure 3 This is the detection of the pH range for the growth of Microbacterium sp. SCSIO 12466.
[0020] Figure 4 This is the detection of the NaCl concentration range for the growth of Microbacterium sp. SCSIO 12466.
[0021] Figure 5 This is the standard curve of heme. Detailed implementation manners
[0022] The following examples are further descriptions of the present invention rather than limitations thereof.
[0023] Example 1 Isolation of Microbacterium sp. SCSIO 12466
[0024] (1) Coral samples were collected from the Luhuitou fringing reef area in Sanya, China (109°29′E, 18°13′N). After collection, the surface of the samples was immediately rinsed with sterile seawater to remove attached sediment and surface plankton. Subsequently, a coral tissue block of about 5 cm 3 was cut, and a tissue suspension was prepared by flushing the coral tissue with an air gun at an appropriate air pressure.
[0025] (2) The obtained suspension was serially diluted 10-fold with sterile seawater (10 -1 to 10 -6 ). 100 μL was aspirated from each dilution and evenly spread on a pre-prepared MA medium plate. The formula of the MA medium includes: 5 g of peptone, 1 g of yeast extract, 0.1 g of ferric citrate, 15 g of agar powder, and 1 L of natural seawater, and the pH value was adjusted to 7.0.
[0026] (3) The coated medium was cultured at 25 °C for 7 days. Single colonies were picked with a sterile inoculation loop and streaked on a new MA medium plate for multiple rounds of purification operations. The purified strain was preserved with a 25% (w / v) glycerol solution and stored in a -80 °C refrigerator for subsequent experiments, obtaining Microbacterium sp. SCSIO 12466.
[0027]
[0028] The strain SCSIO 12466 of the present invention belongs to Microbacterium and is named Microbacterium sp. SCSIO 12466. It was deposited on December 6, 2024, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC), Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, with the deposit number CGMCC No. 32943.
[0029] Example 2 Detection of the adaptability of Microbacterium sp. SCSIO 12466 to temperature, pH value, and NaCl concentration
[0030] 1. Detection of temperature adaptability
[0031] (1) Inoculate the pure culture of Microbacterium SCSIO 12466 into a test tube containing MB liquid medium. The formula of MB liquid medium is: 5 g of peptone, 1 g of yeast extract, 0.1 g of ferric citrate, 30 g of sea salt, dissolved in 1 L of pure water, pH 7, sterilized at 121 °C for 20 minutes.
[0032] (2) Place the test tube described in step (1) in a shaker and culture for 36 hours to obtain the growth conditions of the strain adapted to temperatures of 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, and 45 °C.
[0033] The results are shown in Figure 2 . It can be seen from Figure 2 that the results show that the suitable temperature range for the growth of Microbacterium SCSIO 12466 is 10 °C to 40 °C, and the most suitable growth temperature is 25 °C to 30 °C.
[0034] 2. Detection of pH value adaptability
[0035] (1) Inoculate the pure culture of Microbacterium SCSIO 12466 into a test tube containing MB liquid medium.
[0036] (2) Place the test tube described in step (1) in a shaker and culture at 25 °C for 36 hours to obtain the growth conditions of the strain adapted to pH values of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0.
[0037] The results are shown in Figure 3 . It can be seen from Figure 3 that the suitable pH range for the growth of Microbacterium SCSIO 12466 is 5 to 10, and the most suitable growth pH range is 6 to 7.
[0038] 3. NaCl Concentration Adaptability Detection
[0039] (1) Inoculate the pure culture of Microbacterium sp. SCSIO 12466 into a test tube containing MB liquid medium.
[0040] (2) Place the test tube described in step (1) in a shaker and incubate at 25 °C for 36 hours to obtain the growth conditions of the strain under NaCl with mass percentage concentrations of 1%, 2%, 3%, 4%, 5%, 7%, 10%, and 15%.
[0041] The results are shown in Figure 4 . From Figure 4 it can be seen that the suitable salinity range for the growth of Microbacterium sp. SCSIO 12466 is 1% - 4% NaCl in mass percentage concentration, and the most suitable salinity range for growth is 3% NaCl in mass percentage concentration.
[0042] Example 3 Preparation of Heme by Microbacterium sp. SCSIO 12466
[0043] Inoculate strain SCSIO 12466 into 5 mL of MB liquid medium and incubate at 25 °C and 180 rpm for 3 days until the end of the logarithmic growth phase. Subsequently, transfer the bacterial liquid to 50 mL of fresh MB liquid medium at an inoculation ratio of 1% by volume and continue to incubate at 25 °C and 180 rpm for 3 days. After the incubation, centrifuge the culture solution at 4 °C and 12000 r / min for 5 minutes to collect the cell pellet. Resuspend the cell pellet with 10 mL of sterile deionized water and centrifuge again at 12000 r / min for 5 minutes, discard the supernatant, and record the wet weight of the cells.
[0044] Resuspend 0.52 g of cells with 500 μL of 20 mmol / L oxalic acid solution, transfer the mixture to a 1.5 mL amber centrifuge tube, gently mix and place at 4 °C for 16 h. Subsequently, add 500 μL of 2 mol / L oxalic acid solution to the centrifuge tube, mix well and divide into two groups for treatment: Take 500 μL of the sample and transfer it to another 1.5 mL amber centrifuge tube, heat it in a 95 °C water bath for 30 minutes for the fluorescence detection of the total concentration of porphyrin and heme; the other 500 μL of the sample is placed at room temperature for the fluorescence detection of the porphyrin concentration. After the sample is cooled to room temperature, centrifuge at 12000 r / min for 5 min respectively, take 200 μL of the supernatant and add it to a black transparent-bottom 96-well plate for fluorescence measurement. The detection conditions are excitation wavelength 400 nm and emission wavelength 620 nm.
[0045] To draw the heme standard curve, use 0.25% (w / v) of Na2 CO 3 Prepare standard solutions of hemin with different concentrations. Mix the standard sample with 2 mol / L oxalic acid solution in equal volume ratio of 1:1 and heat in a water bath at 95 °C for 30 min. After the reaction, wait for the sample to cool to room temperature, take 200 μL of the supernatant and add it to a black transparent-bottom 96-well plate, and measure the fluorescence value under the conditions of excitation wavelength 400 nm and emission wavelength 620 nm. According to the test results, plot a standard curve with the fluorescence value as the ordinate and the hemin concentration as the abscissa (see Figure 5 ).
[0046] Substitute the fluorescence value difference of the above experimental sample into the linear regression equation y = 3968.6x + 18395 (where y is the fluorescence value), and calculate the value of x, that is, the hemin concentration in the experimental sample. Subsequently, calculate the amount of hemin contained in each gram of wet bacterial cells based on the wet weight of the bacterial cells used for hemin extraction. The final result shows that the hemin content per gram of wet bacterial cells is 50.56 ± 1.43 μg / g (micrograms per gram of wet bacterial cells).
[0047] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as a limitation of the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention.
Claims
1. A strain of Microbacterium sp. SCSIO 12466, whose deposit number is: CGMCC No.32943.
2. A microbial agent, characterized in that: The invention contains the Microbacterium SCSIO 12466 as claimed in claim 1 as an active ingredient.
3. Use of the Microbacterium SCSIO 12466 described in claim 1 or the microbial agent described in claim 2 in the preparation of heme.
4. A method for producing heme using the Microbacterium SCSIO 12466 of claim 1, characterized in that: The heme is prepared from the fermentation culture of Microbacterium SCSIO 12466CGMCC No.32943.
5. The method according to claim 4, characterized in that The fermentation culture of Microbacterium SCSIO 12466 is obtained by fermenting and culturing Microbacterium SCSIO 12466 using MB liquid culture medium as fermentation medium.
6. The method according to claim 5, characterized in that The MB liquid culture medium comprises: 5 g peptone, 1 g yeast extract, 0.1 g ferric citrate, 1 L seawater, pH 7.
0.
7. The method according to claim 4, characterized in that The fermentation culture is bacterial cells.
8. The method according to claim 5, characterized in that The culture condition is culturing at 25° C. for 36-72 hours.