Streptomycete and application thereof in synthesis of phosphatidylserine

Abstract

The invention relates to streptomyces and application thereof in synthesis of phosphatidylserine, and belongs to the technical field of biology. According to the streptomyces disclosed by the invention, fermentation liquor generated after fermentation takes soybean lecithin and L-serine as raw materials, and phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and the like in lecithin can be simultaneously converted to efficiently generate phosphatidylserine (PS). The name of the streptomyces strain is Streptomyces sp.LN2, and the streptomyces strain is preserved in the China General Microbiological Culture Collection Center on April 1, 2024, and the preservation number of the strain is CGMCC N0.30230.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a streptomyces and application thereof in synthesizing phosphatidylserine. Background Art

[0002] Phospholipids are commonly known as egg cithin. In a narrow sense, they specifically refer to phosphatidylcholine (PC). In a broad sense, they are a general term for a class of mixtures containing phosphatidylglycerides. According to the different phosphatidyl groups contained in phospholipids, they can be divided into: phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidic acid (PA). Among them, phosphatidylserine is the only phospholipid that can regulate the state of key proteins in the cell membrane. It plays an important role in maintaining the structure and function of the cell membrane and can activate the metabolic synthesis of various enzymes. Because PS has a strong lipophilicity, it can quickly pass through the blood-brain barrier into the brain after absorption, soothing vascular smooth muscle cells and increasing blood supply to the brain. It is known as a new emerging "smart nutrient" after choline "brain gold" DHA. Studies have shown that PS has the effects of preventing Alzheimer's disease, improving brain memory, relieving depression, and improving children's attention. It has been listed as a new resource food and functional food, and is therefore widely used in health food, cosmetics, and the pharmaceutical industry. However, the natural PS content is scarce and the extraction process is complicated, which cannot meet market demand. Therefore, it is of great significance to prepare high-purity and high-quality PS products.

[0003] Subsequent studies have found that PS has similar structures to PC and PE and can be converted into each other, so the research on the preparation of functional phospholipids by enzymes has been carried out. The preparation of phosphatidylserine by enzyme catalysis has the characteristics of high product quality, simple operation, and mild process conditions. Therefore, the method of synthesizing functional phospholipids by bioenzyme catalysis is very meaningful and it is easier to obtain products that are difficult to obtain by chemical catalysis.

[0004] At present, the bioenzymatic production of PS mainly uses phospholipase D (PLD) to catalyze the transphosphatidyl reaction of lecithin (derived from soybeans, sunflower seeds, eggs, etc.) and L-serine to produce phosphatidylserine. PLD is ubiquitous in plants, animals and microorganisms, among which microbial PLD has become the most widely used enzyme due to its high catalytic performance and simple purification process. The reported microorganisms that produce phospholipase D mainly include Streptomyces, Corynebacterium, Escherichia coli, Salmonella, Pseudomonad, Vibrio harveyi, Bacillus cereus, Achromobacter, and Aspergillus oryzae. Most of the reported phospholipase D-producing microorganisms use lecithin as a substrate. However, in addition to the main component PC, soybean lecithin also has several important components PE (content of about 15%) and PI (content of about 15%). Nevertheless, these microorganisms can only catalyze the translipidation reaction of PC to generate PS, so it is difficult to obtain high-content PS from low-content lecithin. This leads to a significant decrease in the utilization rate of the raw material lecithin, thereby increasing the production cost. Even if PS is synthesized directly from PC as a raw material, it will still lead to an increase in reaction costs because it is expensive. Therefore, it is necessary to screen a strain that can use lecithin as a raw material and can simultaneously utilize PC and PE to convert into PS, and provide a safe, environmentally friendly, low-cost, and high-utilization method for producing phosphatidylserine, which is of great significance for the industrial preparation of phosphatidylserine. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art. The present invention first provides a PS-producing strain screened from a soil sample, a Streptomyces sp. LN2, which was deposited on April 1, 2024 at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.30230.

[0006] The invention also provides application of the streptomyces in synthesizing phosphatidylserine.

[0007] The application of the present invention is to use the fermentation liquid of the streptomyces as a catalyst, soybean lecithin and L-serine as raw materials, homogenously mix and react, and can simultaneously catalyze the phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the raw materials to synthesize phosphatidylserine.

[0008] When the homogeneous mixing is reacted, the reaction temperature is 40-45° C. and the reaction time is 6-8 hours.

[0009] After the reaction is completed, water is added to the reaction solution to dissolve L-serine, centrifugation is performed to obtain an aqueous phase and a precipitate, the precipitate is washed with water, and centrifugation is performed again to obtain an aqueous phase and a precipitate, the aqueous phases are combined, concentrated by membrane filtration to recover L-serine, and the precipitates are combined and vacuum freeze-dried to obtain a phosphatidylserine product.

[0010] Beneficial effects:

[0011] The invention uses the fermentation liquid of the screened streptomyces as a catalyst and soybean lecithin and L-serine as raw materials, and can simultaneously transform phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in lecithin to efficiently generate phosphatidylserine (PS), thereby improving the utilization rate of raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings further illustrate the present invention, but the contents in the accompanying drawings do not constitute any limitation to the present invention.

[0013] Figure 1 : Colony morphology of the strain Streptomyces sp.LN2 of the present invention cultured on solid state;

[0014] Figure 2 : Phylogenetic tree diagram of strain Streptomyces sp.LN2 of the present invention;

[0015] Figure 3 : The liquid phase detection diagram of the strain Streptomyces sp.LN2 of the present invention used as the raw material for phosphatidylserine synthesis reaction;

[0016] Figure 4 : A liquid phase detection diagram showing the completion of the phosphatidylserine synthesis reaction using the strain Streptomyces sp. LN2 of the present invention.

[0017] Biomaterial deposit information:

[0018] Streptomyces sp. LN2 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 1, 2024, and its deposit number is CGMCC NO.30230. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical solution of the present invention. The reagents or materials used in the embodiments, unless otherwise specified, are all from commercial channels.

[0020] Example 1: Method for determining lipid transfection activity of crude enzyme solution

[0021] (1) Determined by catalyzing PC and L-serine to generate PS:

[0022] Take an appropriate amount of soybean-derived phosphatidylcholine (PC) and dissolve it in 10 mL of chromatographic-grade chloroform (final concentration 5 mg / mL) as the organic phase; take another 10 mL of the fermentation crude enzyme solution, add L-serine (final concentration 50 mg / mL), Triton X-100 (v / v, 2.4%), and CaCl2 (final concentration 80 mM) as the aqueous phase; mix the organic phase and the aqueous phase in a conical flask in a ratio of 1:1, and place it on a constant temperature shaking table at 37°C for 1-8 hours.

[0023] (2) Liquid phase detection method:

[0024] After the reaction, 500ul of the reaction solution sample was taken and centrifuged at 12000rpm for 10min, the upper organic phase was aspirated, and the organic phase was volatilized and dissolved with mobile phase A of n-hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio), and then passed through a 0.45μm organic membrane for HPLC analysis.

[0025] Chromatographic conditions: lichrosphere 100diol (4mm x 125mm) chromatographic column, ELSD evaporative light scattering detector, mobile phase: mobile phase A is n-hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio), mobile phase B is isopropanol-water-acetic acid-triethylamine (850:140:10:0.8, volume ratio); flow rate: 1mL / min; measurement temperature: 30℃; injection volume: 10μL; evaporation temperature: 80℃; nebulization temperature: 50℃; nitrogen flow rate: 1Lmin; gradient elution: according to Table 1.

[0026] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of phosphatidylcholine into 1 μmol of phosphatidylserine per minute at 37°C.

[0027] Table 1 Gradient elution program

[0028] step Running time / min Mobile phase A / (%) Mobile phase B / (%) 1 0 100 0 2 20 50 50 3 25 0 100 4 29 0 100 5 30 100 0 6 35 100 0

[0029] Example 2: Screening and identification of Streptomyces sp. LN2

[0030] According to Bergey's Bacterial Identification Manual, the strain was first determined to be Streptomyces through morphological observation and physiological and biochemical tests. The Streptomyces sp. LN2 of the present invention has typical Streptomyces characteristics: when cultured on solid, the hyphae are branched, without septa, Gram-positive, and the hyphae are intertwined to form small colonies with dense texture, which are dry, opaque, and difficult to pick. The diameter of the colony of this strain can reach 3-4 mm in 5-7 days. The hyphae are white at first, and then light gray and fluffy, such as Figure 1 shown.

[0031] Then, the 16SrDNA cluster analysis method was used to commission General Biotech (Anhui) Co., Ltd. to determine the 16SrDNA sequence of the isolated strain, and the reported 16SrDNA sequence of Streptomyces was downloaded from NCBI. The phylogenetic tree of the 16SrDNA of the strain was obtained through analysis and comparison using the software MEGA10.0. The results are as follows: Figure 2 , showing that the physiological and biochemical results of strain LN2 were consistent with those of Streptomyces and belonged to the genus Streptomyces.

[0032] 16SrDNA sequence determination results:

[0033]

[0034] The specific screening steps of the strain of the present invention are as follows:

[0035] (1) Plate screening: The collected soil samples were processed and cultured at 28°C using lecithin as the substrate and inducer and bromocresol purple as the color developer. The strains that produced white transparent circles around the colonies were the strains that could transform lecithin. Single colonies with transparent circles were picked and cultured on solid plates for 72 hours before rescreening.

[0036] The solid culture medium used was: yeast extract 4 g / L, maltodextrin 10 g / L, glucose 4 g / L, agar 15 g / L, sterilized at 115°C for 20 min.

[0037] (2) Shake flask rescreening: The strains obtained from the initial plate screening were inoculated into the seed culture medium for shake flask culture at 28°C for 5 days to obtain the seed culture solution. The seed culture medium used was: soy peptone 20g / L, maltodextrin 20g / L, glucose 4g / L, K2HP041g / L, MgS04·7H2O 1g / L, CaCl20.1g / L, Na2HPO4·12H2O6g / L, sterilized at 115°C for 20min.

[0038] (3) Fermentation detection of enzyme activity: Then the obtained seed culture liquid is inoculated into the basic fermentation medium for cultivation, the inoculation amount is 2% of the volume of the basic fermentation medium, 28°C, and cultured for 48 hours to obtain the fermentation culture liquid. The basic fermentation medium used is: soy peptone 30g / L, maltodextrin 20g / L, glucose 7g / L, K2HP041g / L, MgS04·7H2O 1g / L, CaCl20.1g / L, Na2HPO4·12H2O 6g / L, sterilized at 115°C for 20min. The fermentation culture liquid is centrifuged at 6000rpm for 30min to remove the Streptomyces cells, and the supernatant is obtained and then ultrafiltered through an ultrafiltration membrane to remove salts and impurities to obtain a crude enzyme liquid. The enzyme activity is determined according to the above enzyme activity determination method, and strains with high lipid transfection activity are screened for the preparation of phosphatidylserine. The results screened out a strain with a maximum translipidation activity of 58.4 U / ml, and named it Streptomyces sp. LN2.

[0039] Example 3: Bacteria fermentation

[0040] The Streptomyces sp.LN2 strain screened in Example 2 was fermented and expanded under the following conditions:

[0041] (1) Plate culture: 20 g / L soy peptone, 20 g / L maltodextrin, 4 g / L glucose, 1 g / L K2HP04, 1 g / L MgS04·7H2O, 0.1 g / L CaCl2, 6 g / L Na2HPO4·12H2O, and 15 g / L agar were sterilized at 115°C for 20 min to obtain a plate culture medium. Streptomyces sp. LN2 was inoculated onto the plate culture medium and cultured at 28°C for 72 h.

[0042] (2) Primary seed liquid culture: Take a 250 mL conical flask, add 6 glass beads, and prepare 60 mL of culture medium: 20 g / L soy peptone, 20 g / L maltodextrin, 4 g / L glucose, 1 g / L K2HP04, 1 g / L MgS04·7H2O, 0.1 g / L CaCl2, 6 g / L Na2HPO4·12H2O, and sterilize at 115°C for 20 min. Pick several large colonies of Streptomyces sp. LN2 obtained in step (1), inoculate them into the primary seed culture medium, and culture at 200 rpm, 28°C for 48 h.

[0043] (3) Secondary seed culture: Take a 2L conical flask, add 8 glass beads, and prepare 300mL culture medium: 30g / L soy peptone, 20g / L maltodextrin, 4g / L glucose, 1g / L K2HP04, 1g / L MgS04·7H2O, 0.1g / L CaCl2, 6g / L Na2HPO4·12H2O, sterilize at 115°C for 20min. Add all the primary seed culture medium obtained in step (2), culture at 200rpm, 28°C, and culture for 48h.

[0044] (4) Fermentation broth culture: A 5L fermentation tank was used to prepare 3L fermentation medium: 30 g / L soy peptone, 20 g / L maltodextrin, 7 g / L glucose, 1 g / L K2HP04, 1 g / L MgS04·7H2O, 0.1 g / L CaCl2, 6 g / L Na2HPO4·12H2O, and 1 mL / L defoamer, and sterilized at 115°C for 20 min. The secondary seed culture obtained in step (3) was inoculated into the fermentation medium, with an initial stirring speed of 200 rpm, a ventilation volume of 1 vvm, 28°C, and a culture time of 48 h to obtain a fermentation broth.

[0045] (5) Centrifugal ultrafiltration to obtain crude enzyme solution: The fermentation culture solution obtained in step (4) is allowed to stand for a period of time, and then centrifuged at 6000 rpm for 30 min to remove the Streptomyces sp. LN2 bacteria, and the supernatant is then ultrafiltered through an ultrafiltration membrane to remove salts and foreign proteins to obtain a crude enzyme solution.

[0046] Example 4: Preparation of phosphatidylserine

[0047] Take an appropriate amount of the fermentation crude enzyme solution in Example 3 above, prepare 270 ml of an enzyme-containing aqueous solution, the enzyme activity of the enzyme solution is 10 U / mL, and the pH is adjusted to 7.0; add 50 g of soybean lecithin raw material (phosphatidylcholine content is 44.33%) and 40 g of L-serine to the enzyme-containing aqueous solution, stir uniformly, and react at 40-45° C. for 6-8 hours;

[0048] After the reaction, 500ul of the reaction solution sample was taken and centrifuged at 12000rpm for 10min, the upper organic phase was aspirated, and the organic phase was volatilized and dissolved with mobile phase A of n-hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio), and then passed through a 0.45μm organic membrane for HPLC analysis.

[0049] Next, 270 ml of water was added to the reaction solution, the serine was dissolved by stirring, and the aqueous phase was separated by centrifugation to obtain 90 g of centrifugal precipitate; the centrifugal precipitate was washed with 450 ml of pure water, the aqueous phase was separated by centrifugation, and about 990 ml of the aqueous phase separated by two centrifugations was filtered and concentrated to 150 ml by membrane (reverse osmosis membrane) to obtain an L-serine aqueous solution for the next batch reaction, and 90 g of the second centrifugal precipitate was obtained; the two centrifugal precipitates were subjected to vacuum freeze drying to obtain 43 g of a product containing high-purity phosphatidylserine.

[0050] The reaction raw material soybean lecithin and the liquid phase detection results at the end of the reaction are as follows Figure 3-4 As shown: Figure 3 The substances detected from the reaction raw materials are, according to the peak time, PA retention time 4.553min, PE retention time 8.012min, PC retention time 8.755min, PI retention time 10.873min; Figure 4 The substances detected at the end of the reaction are, in order of peak elution time, PA retention time 4.276min, PE retention time 7.879min, PC retention time 8.599min, the new peak, i.e., product PS retention time 10.192min, PI retention time 10.715min; the percentage content of each component in the reactant is shown in Table 2, and the results show that the Streptomyces sp.LN2 of the present invention can not only convert the main component PC in the substrate lecithin into PS, but also the contents of PE and PI are significantly reduced, indicating that the Streptomyces sp.LN2 also has the activity of converting PE and PI to generate PS, so that the raw materials are fully utilized, and the PC conversion rate is as high as 109.77%.

[0051] Table 2 Content of each component in the reactants

[0052]

[0053] Note: PC conversion rate = product PS content ÷ raw material PC content x 100%

[0054] Example 5: Verification of the activity of Streptomyces sp. LN2 in converting PE to PS

[0055] Take an appropriate amount of phosphatidylethanolamine (PE) dissolved in 10 mL of chromatographic pure chloroform (final concentration 5 mg / mL) as the organic phase; take another 10 mL of the fermentation crude enzyme solution, add L-serine (final concentration 50 mg / mL), Triton X-100 (v / v, 2.4%), and CaCl2 (final concentration 80 mM) as the aqueous phase; mix the organic phase and the aqueous phase in a conical flask in a ratio of 1:1, and place it on a constant temperature shaking shaker at 37°C for 1-8 hours.

[0056] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of phosphatidylethanolamine into 1 μmol of phosphatidylserine per minute at 37°C.

[0057] The content of phosphatidylserine was determined according to the sample treatment method and HPLC-ELSD detection method in Example 1, and the enzyme activity was calculated. It was found that the activity of Streptomyces sp. LN2 in converting PE to PS was 33.7 U / ml.

[0058] Example 6: Verification of the activity of Streptomyces sp. LN2 transforming PI to produce PS

[0059] Take an appropriate amount of phosphatidylinositol (PI) dissolved in 10 mL of chromatographic pure chloroform (final concentration 5 mg / mL) as the organic phase; take another 10 mL of the fermentation crude enzyme solution, add L-serine (final concentration 50 mg / mL), Triton X-100 (v / v, 2.4%), and CaCl2 (final concentration 80 mM) as the aqueous phase; mix the organic phase and the aqueous phase in a conical flask in a 1:1 ratio, and place it on a constant temperature shaking table at 37°C for 1-8 hours.

[0060] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of phosphatidylinositol into 1 μmol of phosphatidylserine per minute at 37°C.

[0061] The content of phosphatidylserine was determined according to the sample treatment method and HPLC-ELSD detection method in Example 1, and the enzyme activity was calculated. It was found that the activity of Streptomyces sp. LN2 in converting PI to PS was 12.6 U / ml.

[0062] The technical principle of the present invention is described above in conjunction with specific embodiments. These descriptions are only for explaining the principle of the present invention and cannot be interpreted as limiting the scope of protection of the present invention in any way. Based on the explanations herein, those skilled in the art can associate other specific embodiments of the present invention without creative work, and these equivalent variations or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A Streptomyces, characterized in that The deposit number of the Streptomyces sp. LN2 is CGMCCNO.30230.

2. Use of the Streptomyces described in claim 1 in synthesizing phosphatidylserine.

3. The use according to claim 2, characterized in that: The fermentation liquid of the streptomyces is used as a catalyst, and soybean lecithin and L-serine are used as raw materials, and the mixture is homogeneously mixed for reaction, thereby catalyzing phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the raw materials to synthesize phosphatidylserine at the same time.

4. The use according to claim 3, characterized in that: When the mixture is homogeneously mixed for reaction, the reaction temperature is 40°C to 45°C and the reaction time is 6 to 8 hours.

5. The use according to claim 3, characterized in that: After the reaction is completed, water is added to the reaction solution to dissolve L-serine, and centrifugation is performed to obtain an aqueous phase and a precipitate. The precipitate is washed with water, and centrifugation is performed again to obtain an aqueous phase and a precipitate. The aqueous phases are combined and concentrated by membrane filtration to recover L-serine, and the precipitates are combined and vacuum freeze-dried to obtain a phosphatidylserine product.

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