Application of Enterococcus monteri in producing dihydroouabain
By using Enterococcus mondtii E14 strain to shake culture in liquid LB culture medium, the shortcomings of the dihydro-owain synthesis pathway were successfully solved, efficient synthesis and simplified extraction process, and a new biopharmaceutical pathway was provided.
Patent Information
- Application Number
- CN202410722706.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-05
- Publication Date
- 2025-03-18
- Estimated Expiration
- 2044-06-05
AI Technical Summary
The prior art is difficult to produce high-content dihydro-uabain on a large scale, and its content is low in synthesis in mammals and plants, and the conditions for isolation, extraction and purification are complex.
A new synthetic pathway was provided by shaking culture in liquid LB culture medium using Enterococcus mondtii E14 strain, providing a new synthetic pathway.
It realizes efficient synthesis of dihydro-owabain, simplifies the extraction process, is easy to cultivate on a large scale indoor, and has the advantages of convenience, efficiency and simplicity.
Smart Images
Figure CN118421745B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to application of Enterococcus monteri in producing dihydroouabain, and belongs to the technical field of microorganisms. Background Art
[0002] Dihydroouabain is a type of cardiac glycoside, belonging to the subclass of steroidal saponins, also known as dihydroouabain (curare) glycoside. It is mainly used in the medical field and is a type of drug that selectively acts on the heart. Studies have shown that dihydroouabain has a bidirectional effect on endothelial cells. It participates in the regulation of cell proliferation under physiological conditions, and causes heterozygous cell death and mediates vascular remodeling under pathological conditions. It has a direct electrophysiological effect on cardiomyocytes. In addition, dihydroouabain is a Na + -K + -ATPase inhibitor, which enhances the radiosensitivity of cervical cancer cells and eliminates the radiation-induced S phase arrest in cervical cancer cells, is a new type of radiosensitizer for the treatment of cervical cancer. However, dihydroouabain is currently only synthesized in mammals and plants, but its content is low, only a few nanograms, and in mammalian and plant cultures, the conditions for separating, extracting and purifying dihydroouabain are complex, making it difficult to produce dihydroouabain on a large scale.
[0003] This study used a method for producing dihydroouabain by microorganisms, which increased the synthesis pathway of dihydroouabain and improved its synthesis capacity, and can be effectively used for the synthesis of dihydroouabain. Summary of the invention
[0004] The present invention provides an application of Enterococcus monteri in the production of dihydroouabain, and the specific application method is:
[0005] (1) Inoculate the Enterococcus monnieri strain stored in glycerol into liquid LB culture medium and place it on a shaker for shaking culture to obtain an Enterococcus monnieri seed liquid.
[0006] (2) Inoculate 5% of the seed solution obtained in step (2) into a new liquid LB culture medium and shake culture the medium to obtain a fermentation liquid containing dihydroouabain.
[0007] Preferably, the Enterococcus mundtii is Enterococcus mundtii E14, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on January 4, 2024, with a deposit number of GDMCC No: 64240, and an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0008] Preferably, in step (2), the Enterococcus mongoliae seed solution is inoculated at an inoculation rate of 5%.
[0009] Preferably, in step (1) and step (2), the seed solution is placed in a shaker for shaking culture, the temperature of the shaker is 30°C, and the rotation speed is 180 rpm; wherein the OD of the seed solution in step (1) is 600nm =1.0; the culture time in step (2) is 72 hours.
[0010] Beneficial Effects of the Invention
[0011] (1) The present invention is the first to use Enterococcus monteri to synthesize dihydroouabain, opening up a new approach for the synthesis of dihydroouabain.
[0012] (2) The strain of Enterococcus monteroi is simple to culture, grows quickly, is easy to extract, and is easy to culture and reproduce in large quantities indoors and use. Therefore, the synthesis of dihydroouabain using Enterococcus monteroi has the advantages of being convenient, efficient, and simple. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 This is a colony morphology diagram of Enterococcus mundtii E14 in Example 1.
[0014] Figure 2 This is a picture of Enterococcus mundtii E14 after Gram staining in Example 1 under an electron microscope at 100 times magnification.
[0015] Figure 3 This is the phylogenetic tree of Enterococcus mundtii E14 in Example 1.
[0016] Figure 4 The figure is a comparison of the content of dihydroouabain in different strains in Example 3. DETAILED DESCRIPTION
[0017] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.
[0018] Solid LB medium: 5 g yeast extract, 10 g peptone, 5 g NaCl, 15-20 g agar, 1 L water, pH 7.4-7.6, 121°C, sterilize for 30 minutes.
[0019] Liquid LB medium: 5 g yeast extract, 10 g peptone, 5 g NaCl, 1 L water, pH 7.4-7.6, 121°C, sterilization for 30 minutes.
[0020] Unless otherwise specified, the equipment used in the present invention are all commonly used equipment in the field, and the drugs and reagents used are all conventionally commercially available analytical grade.
[0021] Example 1
[0022] Isolation and Identification of Enterococcus mundtii E14 Strain
[0023] The Enterococcus mundtii E14 is isolated and purified from the midgut of tomato leafminer larvae, specifically comprising the following steps:
[0024] (1) Collect healthy second-instar larvae of tomato leafminer and freeze them immediately on ice.
[0025] (2) Disinfect the larvae of tomato leafminer in 75% alcohol for 90 seconds, and finally rinse them with sterile water three times, each time for 1 minute.
[0026] (3) After cleaning, place it in a sterile, clean culture dish, use a sterile insect pin to remove the intestine from the tail of the insect body, and place it in a sterile centrifuge tube (1.5 mL) containing 1 mL of sterile phosphate buffered saline (1× PBS, pH 7.4) buffer for bacterial isolation.
[0027] (4) Use LB medium and the plate spreading method for inoculation. Incubate the inoculated plates at 30°C for 1 week.
[0028] (5) The growing bacteria in the LB medium in step (4) were isolated and purified by the plate streak method. The colonies were purified twice on LB solid medium, and the pure cultured bacterial strains were preserved by glycerol freezing for further molecular and morphological identification. A single colony was picked from the second purified medium and placed in 1 mL of LB liquid medium and shaken at 180 rpm and 30°C for 12 h. The cultured bacterial liquid was sent to Qingke Biotechnology Co., Ltd. for sequencing (using universal primers 27F and 1492R to amplify the 16S rDNA gene for sequencing). The 16S rDNA sequence is shown in SEQ ID No. 1. The sequencing results were spliced using Contigexpress software and compared with the GenBank database to identify the isolated bacteria. A phylogenetic tree was constructed based on the 16S rDNA gene of the strain using MEGA6.5 and the neighbor-joining method, as shown in Figure 1. Figure 3 As shown, a strain of Enterococcus mundtii was isolated and named Enterococcus mundtii E14.
[0029] The Enterococcus mundtii E14 strain obtained by screening has the following characteristics:
[0030] (1) The strain Enterococcus mundtii E14 obtained by screening was streaked on LB medium and cultured at 30°C for 48 hours. It was found that the colonies were round or oval yellow, with a smooth and shiny surface and arranged in chains, such as Figure 1 shown.
[0031] (2) After Gram staining, the strain was observed under a 100x electron microscope and found to be Gram-positive. The Monterey cells were rod-shaped, such as Figure 2 shown.
[0032] Example 2
[0033] Application of Enterococcus mundtii E14 in the production of dihydroouabain
[0034] (1) Inoculate 50 μL of Enterococcus mundtii E14 preserved in glycerol into 1 mL of LB liquid culture medium, place in a shaker at 30°C and 180 rpm for 12 h, and make an OD 600 =1.0 seed solution.
[0035] (2) Add 5% of the seed solution after culture into 100 mL of LB liquid medium and shake at 180 rpm and 30°C for 72 h to obtain a fermentation broth containing dihydroouabain.
[0036] The purification method of dihydroouabain fermentation broth is as follows:
[0037] (1) Centrifugation of fermentation broth: After fermentation, the fermentation broth in the conical flask was transferred to 800 mL centrifuge cups in batches. Ethyl acetate (EtOAc) and methanol (MeOH) were used to dissolve the broth during the transfer process. Each centrifuge cup was filled with about 1 / 2 of the sample. After weighing and balancing on a balance, the cups were placed opposite each other in a refrigerated centrifuge for centrifugation (6500 r / min, 20 min).
[0038] (2) Fermentation broth extraction: After centrifugation, transfer the supernatant to a 10L beaker, add 5L of supernatant and an equal volume of EtOAc to each beaker, seal with a sealing film and place in an ultrasonic cleaner for 1h of ultrasonic extraction, then transfer to a fume hood for overnight extraction. After the extraction, use a separatory funnel to remove the EtOAc portion of the upper layer and add it to a 500mL rotary evaporator for rotary evaporation concentration (water bath 40°C, cold well -20°C), continue to add supernatant, recycle the distilled EtOAc, and repeat three times. After the remaining bacterial cells are collected, soak them in MeOH for 3d, ultrasonically extract them, and add the extract to the rotary evaporator. The organic phase is concentrated under reduced pressure to obtain an extract and transferred to a sample bottle for low-temperature storage (below 10°C) for later use.
[0039] Example 3
[0040] Comparison of common Enterococcus mundtii and Enterococcus mundtii E14 of the present invention in the synthesis of dihydroouabain, the specific method is:
[0041] (1) Common Enterococcus mundtii and the Enterococcus mundtii E14 of the present invention were cultured according to the method described in Example 2 to obtain culture fluid.
[0042] (2) The culture solution was centrifuged at 13,000 rpm for 20 min, the supernatant was collected, and the filtrate was filtered through a 0.22 pm microporous membrane. 1 mL of the filtrate was taken for measurement of dihydroouabain content. Each treatment had three biological replicates.
[0043] (3) The sample (filtrate) was analyzed by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MSMS) using a liquid chromatography instrument (UPLC) column (2.1×100 mm, 1.7 μm). The analysis was performed in gradient mode using a mobile phase consisting of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) in a ratio of 20:80. Flow rate: 0.3 mL / min; temperature: 40°C; sample volume: 20 μL. Under this chromatographic condition, the concentration of dihydroouabain in the sample was quantified by comparing the peak area of the sample with the peak area of its respective internal standard. In order to prove that this function does not exist in other strains, the model strain of Enterococcus monteri was selected as a control, and the content of dihydroouabain in the culture medium under different strain treatments was compared, such as Figure 4 shown.
[0044] The results showed that Enterococcus mundtii E14 strain could synthesize dihydroouabain, while common Enterococcus mundtii could not.
Claims
1. An application of Enterococcus mundtii in the production of dihydroouabain, wherein the Enterococcus mundtii is Enterococcus mundtii E14, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on January 4, 2024, with a deposit number of GDMCC No: 64240, and an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. The use according to claim 1, characterized in that: The specific application method is: (1) inoculating the Enterococcus monnieri strain stored in glycerol into LB medium and culturing with shaking to obtain an Enterococcus monnieri seed solution; (2) The Enterococcus monnieri seed solution obtained in step (1) is inoculated into a new LB medium and cultured by shaking to obtain a fermentation solution containing dihydroouabain.
3. The use according to claim 2, characterized in that: In step (2), the Enterococcus mongoliae seed solution is inoculated at an inoculation rate of 5%.
4. The use according to claim 2, characterized in that: In step (1) and step (2), the culture is carried out in a shaker with a shaking temperature of 30° C. and a rotation speed of 180 rpm; wherein the OD600nm of the seed solution in step (1) is 1.0; and the culture time in step (2) is 72 hours.