Pseudomonas fluorescens strain STR2 and its use
Lipases were prepared by screening for Pseudomonas fluorescens STR2 and applied to the hydrolysis of tocopherol succinate, overcoming the shortcomings of traditional catalytic methods and achieving efficient and environmentally friendly tocopherol production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QUFU NORMAL UNIV
- Filing Date
- 2026-04-09
- Publication Date
- 2026-06-02
Smart Images
Figure CN122128174A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a fluorescent Pseudomonas STR2 strain and its applications. Background Technology
[0002] Tocopherols are important natural antioxidants with wide applications in food preservation, feed additives, and pharmaceuticals. With the increasing popularity of green manufacturing concepts, developing safe, efficient, and environmentally friendly tocopherol production processes has become a focus of industry attention.
[0003] Traditional chemical catalysis methods have many drawbacks: catalysts are difficult to recover, reaction conditions are harsh, large amounts of saline wastewater are generated, and harmful byproducts may remain in the product, affecting product quality. In contrast, lipase catalysis has advantages such as mild reaction conditions, high catalytic efficiency, fewer byproducts, and environmental friendliness, aligning with the development direction of green chemistry.
[0004] Microbial lipases are abundant, with *Pseudomonas* being one of the genera with strong lipase-producing capabilities. *Pseudomonas fluorescens*, a representative species of this genus, exhibits excellent catalytic performance in various ester hydrolysis reactions. However, the application of *Pseudomonas fluorescens* in the hydrolysis of tocopherol succinate remains a blank area. Summary of the Invention
[0005] To address the aforementioned problems in the existing technology, the first technical problem to be solved by this invention is to provide a strain of *Pseudomonas fluorescens* STR2 capable of efficiently catalyzing the hydrolysis of tocopherol succinate. The second technical problem to be solved by this invention is to provide specific applications of the aforementioned strain.
[0006] To achieve the above objectives, the technical solution of this application is as follows:
[0007] A strain of *Pseudomonas fluorescens*, classified as Pseudomonas fluorescens, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025606, on March 27, 2025, at Wuhan University, Wuhan, China.
[0008] A lipase, said lipase being prepared by fermentation of the fluorescent Pseudomonas STR2.
[0009] The application of the aforementioned *Pseudomonas fluorescens* STR2, or the aforementioned lipase, in the catalytic hydrolysis of tocopherol succinate to prepare tocopherol.
[0010] In some embodiments, the application specifically includes: adding the crude enzyme solution obtained by fermenting the fluorescent Pseudomonas STR2, or the lipase extracted from the crude enzyme solution, to a reaction system containing tocopherol succinate to carry out a hydrolysis reaction to prepare tocopherol.
[0011] In some embodiments, the reaction system comprises tocopherol succinate, an organic solvent, and the crude enzyme solution or the lipase.
[0012] In some embodiments, the organic solvent is n-hexane.
[0013] In some embodiments, the conditions for the hydrolysis reaction are: reaction temperature 25-40℃, reaction time 6-12 hours, and shaking speed 150-250 r / min;
[0014] A method for preparing tocopherol using the fluorescent Pseudomonas STR2 includes the following steps:
[0015] (1) The fluorescent Pseudomonas STR2 was fermented to obtain a fermentation broth containing lipase. The fermentation broth was centrifuged to obtain a crude enzyme solution.
[0016] (2) The crude enzyme solution obtained in step (1) is mixed with a substrate solution containing tocopherol succinate, and the hydrolysis reaction is carried out under the hydrolysis reaction conditions described above.
[0017] (3) After the hydrolysis reaction is completed, the reaction solution is separated and purified to obtain tocopherol.
[0018] In some embodiments, the fermentation culture medium used in step (1) contains carbon source, nitrogen source, inorganic salts and lipids, and has a pH of 7.0 to 7.2.
[0019] In some embodiments, the carbon source is selected from sugars, the nitrogen source is selected from at least one of ammonium salts and peptones, the inorganic salt is selected from at least one of phosphates and sulfates, and the lipids are selected from vegetable oils.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] This invention screened and obtained a strain of Pseudomonas fluorescens STR2 with high lipase activity. The lipase produced by this strain can efficiently catalyze the hydrolysis of tocopherol succinate to produce tocopherol. Experimental results showed that after 9 hours of catalytic reaction using the crude enzyme solution of this strain, the tocopherol content in the product was increased by 700% compared with the control group. The strain and method provided by this invention have the advantages of high catalytic efficiency, mild reaction conditions, and environmental friendliness, and can effectively replace traditional chemical catalysis processes, making them suitable for the green industrial production of tocopherol. Attached Figure Description
[0022] Figure 1 Figure showing the results of streak culture of STR2 strain;
[0023] Figure 2 The image shows the results of Gram staining of STR2 strain under an oil immersion microscope.
[0024] Figure 3 The phylogenetic tree analysis results of strain STR2 based on the 16S rDNA sequence are shown in the figure.
[0025] Figure 4 The image shows the lipase activity assay results for strain STR2. Detailed Implementation
[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0027] Preservation of biological materials
[0028] The *Pseudomonas fluorescens* STR2 strain involved in this invention was deposited on March 27, 2025, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC No: M 2025606, and the deposit address is Wuhan University, Wuhan, China.
[0029] Culture medium formulation
[0030] Enrichment medium: 6g yeast extract, 1g Na2HPO4, 2g KH2PO4, 1g MgSO4·7H2O, 5g (NH4)2SO4, 0.5g NaCl, 10mL soybean oil, 1000mL H2O, pH 7.0~7.2, autoclaved at 121℃ for 15min.
[0031] Primary screening medium A (neutral red oil plate): 10g peptone, 5g beef extract, 5g NaCl, 10mL soybean oil, 1mL 1.6% neutral red aqueous solution, 20g agar, 1000mL water, pH 7.0-7.2, sterilized at 121℃ for 15min.
[0032] Primary screening medium B (tributyric acid glyceride plate): 10g peptone, 5g yeast powder, 10g NaCl, 2mL tributyric acid glyceride, 20g agar, 1000mL water, pH 7.0~7.2, sterilized at 121℃ for 15min.
[0033] LB slant culture medium: 10g peptone, 5g yeast extract, 10g sodium chloride, 20g agar powder, 1000mL water, pH 7.0~7.2, sterilize at 121℃ for 15min.
[0034] Seed culture medium: 20 g glucose, 5 g (NH4)2SO4, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 25 g peptone, 10 mL soybean oil, 1000 mL water, pH 7.0~7.2, sterilized at 115℃ for 20 min.
[0035] Fermentation medium: 5 g glucose, 1 g (NH4)2SO4, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 20 g peptone, 10 mL soybean oil, pH 7.0~7.2, sterilized at 115℃ for 20 min.
[0036] Chromatographic conditions
[0037] Chromatographic column: Hypersil ODS2 C18 column (5µm, 150×4.6mm); mobile phase: methanol: glacial acetic acid = 1000: 5; flow rate: 1.0mL / min; UV detection wavelength: 284nm; column temperature: 25℃; injection volume: 20µL.
[0038] Soil sample source
[0039] The samples were taken from soil contaminated with grease near the canteen of Qufu Normal University.
[0040] Example 1: Isolation, screening and identification of lipase-producing strains
[0041] 1. Isolation, purification, and primary screening of enzyme-producing bacteria
[0042] Accurately weigh 10g of the collected soil sample and place it in a sterile container containing 90mL of sterile physiological saline. Add an appropriate amount of sterile glass beads, shake thoroughly at room temperature, and let stand for 0.5h. Take 3mL of the supernatant and transfer it to a sterile conical flask containing 27mL of enrichment medium. Incubate at 30℃ and 200r / min with shaking for 24h to allow microbial cell growth and enrichment. Let stand for 20min to obtain the 10g enrichment medium. -2 The diluent; then, using a 1000μL sterile pipette tip, aspirate 10... -2 Transfer 1 mL of diluent to a test tube containing 9 mL of sterile water, and mix the bacterial suspension thoroughly by blowing and aspirating. This yields 10 mL of the solution. -3 Diluent; then use a sterile pipette tip to draw 10... -3 Transfer 1 mL of diluent to a test tube containing 9 mL of sterile water, and mix well by pipetting. This will yield 10 mL of diluent. -4 Diluent; and so on, continuously dilute to prepare 10. -5 10 -6 10 -7 A series of diluted bacterial solutions were prepared.
[0043] Take 200 µL of each serially diluted bacterial suspension and spread it onto primary screening medium A (neutral red oil plate). Use a sterile spreader to evenly spread the suspension on the plate, with three replicates for each dilution (one sterile spreader per dilution). Incubate at 25°C for 48 h. Based on the characteristic red color around the colony, pick single red colonies and purify them on primary screening medium A using the three-zone streak method, then incubate at 25°C for 48 h.
[0044] The isolated and purified bacteria were inoculated onto primary screening medium B (tributyric acid glyceride plates). One type of bacteria was inoculated in the center of each tributyric acid glyceride plate (three replicates for each bacteria). The plates were incubated at 25°C for 48 hours. Colony characteristics were observed, and bacteria that produced hydrolysis zones were selected. The diameter of the clear zone (D) and the colony diameter (d) were measured, and the HC value (HC = D / d) was calculated. Strains with higher HC values were inoculated onto LB slant medium for preservation.
[0045] 2. Rescreening and identification of enzyme-producing bacteria
[0046] The strains obtained from the initial screening were selected, and single colonies were inoculated into 30 mL of seed culture medium and cultured at 30℃ and 200 rpm for 12 h to prepare the seed culture. The seed culture was then transferred to 30 mL of fermentation culture medium at a 1% inoculation rate and cultured at 30℃ and 200 rpm for 48 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was collected as the crude enzyme solution for determining lipase activity. The p-nitrophenol method was used to determine the lipase activity.
[0047] After two rounds of screening, including primary and secondary screening, a strain with high lipase production capacity was obtained and designated STR2. This strain produced an HC value of 4.23 on tributylate plates, with a lipase activity of 149.72 U / mL.
[0048] Strawberry strain STR2 was streaked onto LB agar and incubated at 25°C until single colonies appeared. Colony morphology was then observed. Figure 1 As shown, the colonies are pale yellow, round, opaque, with a smooth, raised surface and neat edges.
[0049] A small amount of fresh bacterial cells was picked up with a sterile toothpick and evenly spread onto a glass slide with a drop of physiological saline. The strain was then Gram-stained, and the staining results were observed using an oil immersion microscope. Figure 2 As shown, the Gram staining result was negative, and the bacterial cell shape was rod-shaped.
[0050] The 16S rDNA sequence of the strain was determined, and the sequence is shown in SEQ ID NO.1. BLAST analysis of the sequencing results was performed on NCBI, and a phylogenetic tree was constructed using MEGA 7.0. The phylogenetic tree constructed from the sequencing results by BLAST analysis is shown below. Figure 3 As shown, the strain was identified as *Pseudomonas fluorescens*, and its physiological and biochemical characteristics are shown in Table 1.
[0051] Table 1 Physiological and biochemical characteristics of STR2 strain
[0052]
[0053] 3. The role of crude enzyme solution in catalyzing the hydrolysis of tocopherol succinate.
[0054] Reaction system: In a 250 mL Erlenmeyer flask, add 20 g of tocopherol succinate and 100 mL of n-hexane, mix well, and then add 30 mL of the crude enzyme solution prepared above. The control group was prepared by replacing the crude enzyme solution with an equal volume of sterile water.
[0055] Reaction conditions: Temperature 37℃, shaking speed 200 r / min, reaction time 9 h. High-performance liquid chromatography (HPLC) was used to detect the tocopherol content in the reaction product, thereby determining the catalytic effect of lipase on the hydrolysis of tocopherol succinate.
[0056] After the reaction, the content of tocopherol in the reaction product was determined by high performance liquid chromatography (HPLC) to evaluate the catalytic effect of lipase on the hydrolysis of tocopherol succinate. Chromatographic conditions: Hypersil ODS2 C18 column (5µm, 150×4.6mm); mobile phase: methanol: glacial acetic acid = 1000: 5; flow rate: 1.0 mL / min; UV detection wavelength: 284 nm; column temperature: 25℃; injection volume: 20µL.
[0057] Test results as follows Figure 4 As shown, the tocopherol content in the product after adding crude enzyme solution increased by 700% compared with the control group, indicating that the lipase produced by strain STR2 has a highly efficient hydrolytic catalytic effect on tocopherol succinate.
[0058] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A strain of fluorescent Pseudomonas STR2, characterized in that, Its classification name is Pseudomonas fluorescensSTR2. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025606, deposit date of March 27, 2025, and deposit address at Wuhan University, Wuhan, China.
2. A lipase, characterized in that, The lipase was prepared by fermentation of the fluorescent Pseudomonas STR2 as described in claim 1.
3. The application of the fluorescent Pseudomonas STR2 of claim 1, or the lipase of claim 2, in the catalytic hydrolysis of tocopherol succinate to prepare tocopherol.
4. The application according to claim 3, characterized in that, Specifically, it includes: The crude enzyme solution obtained by fermentation culture of Pseudomonas fluorescens STR2 as described in claim 1, or the lipase isolated and extracted from the crude enzyme solution, is added to a reaction system containing tocopherol succinate to carry out a hydrolysis reaction to prepare tocopherol.
5. The application according to claim 4, characterized in that, The reaction system consists of tocopherol succinate, an organic solvent, and the crude enzyme solution or the lipase.
6. The application according to claim 5, characterized in that, The organic solvent is n-hexane.
7. The application according to claim 5, characterized in that, The conditions for the hydrolysis reaction are: reaction temperature 25-40℃, reaction time 6-12 hours, and shaking speed 150-250 r / min.
8. A method for preparing tocopherol using the fluorescent Pseudomonas STR2 as described in claim 1, characterized in that, Includes the following steps: (1) The fluorescent Pseudomonas STR2 of claim 1 is fermented to obtain a fermentation broth containing lipase, and the fermentation broth is centrifuged to obtain a crude enzyme solution; (2) The crude enzyme solution obtained in step (1) is mixed with a substrate solution containing tocopherol succinate and subjected to hydrolysis. (3) After the hydrolysis reaction is completed, the reaction solution is separated and purified to obtain tocopherol.
9. The method according to claim 8, characterized in that, The fermentation culture medium used in step (1) contains carbon source, nitrogen source, inorganic salts and lipids, and has a pH of 7.0 to 7.
2.
10. The method according to claim 9, characterized in that, The carbon source is selected from sugars, the nitrogen source is selected from at least one of ammonium salts and peptones, the inorganic salt is selected from at least one of phosphates and sulfates, and the lipids are selected from vegetable oils.