Asian Pseudomonas aeruginosa YHDXY-01 formulation for creatinine degradation and its application
Liquid or solid formulations were prepared by fermentation and enzyme extraction of Pseudomonas Asiana YHDXY-01 strain, which solved the problem of insignificant effect in reducing serum creatinine in existing technologies and achieved the effect of highly efficient biodegradation of creatinine.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING YIRAN BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-05-13
- Publication Date
- 2026-06-30
AI Technical Summary
Existing drugs for lowering human serum creatinine levels are not very effective and have toxic side effects, and there is a lack of highly effective treatment methods.
A strain of Pseudomonas Asiana YHDXY-01 and its preparation are provided. Liquid or solid preparations are prepared by fermentation culture and enzyme extraction, and the bacterial cells and crude enzymes are used to efficiently biodegrade creatinine.
It achieves efficient biodegradation of creatinine, which has important application prospects. It can significantly reduce serum creatinine levels and prevent high serum creatinine and related complications.
Smart Images
Figure CN122303101A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a Pseudomonas aeruginosa YHDXY-01 preparation for biodegrading creatinine, its preparation method and application. Background Technology
[0002] Currently, there are approximately 850 million people worldwide suffering from kidney disease, including those with chronic kidney disease, acute kidney disease, and kidney failure, representing more than 10% of the global population. When kidney function is impaired, metabolic waste products such as creatinine, urea, and uric acid cannot be effectively excreted, leading to a range of health problems, including gastrointestinal disorders, electrolyte imbalances, cardiovascular disease, anemia, and bone diseases, and accelerating kidney damage.
[0003] Creatinine (also known as creatine anhydride, molecular formula: C4H7N3O, molecular weight: 113.1) is a compound formed during muscle energy production, produced by the metabolism of phosphocreatine. The normal range of serum creatinine varies slightly depending on age and sex, but when serum creatinine is significantly higher than the normal range, for example, serum creatinine > 200 μmol / L, approximately 2.3 mg of creatinine per 100 mL of blood, it indicates significant kidney dysfunction, possibly related to acute kidney injury or chronic kidney disease. Excessively high creatinine levels can lead to complications such as hyperkalemia, hyperuricemia, hyperlipidemia, hypoproteinemia, and metabolic acidosis.
[0004] Globally, approximately 1.2 million people die from kidney disease each year, and this number continues to rise, making it a global public health problem. Patients with end-stage renal disease rely on dialysis or kidney transplantation to sustain their lives, placing a heavy burden on their families and society. However, apart from dialysis, there is currently a lack of highly effective treatments in clinical practice to reduce the concentration of metabolic waste products such as serum creatinine. Therefore, there is a need to develop effective therapeutic agents or methods to lower serum creatinine levels. Summary of the Invention
[0005] One of the objectives of this invention is to address the problem that existing drugs for lowering human serum creatinine levels are not very effective and have toxic side effects. This invention provides a strain of Pseudomonas aeruginosa YHDXY-01 that degrades creatinine. The bacterial cells of this strain and the enzymes it produces can efficiently biodegrade creatinine, and it has important application prospects in the removal of creatinine.
[0006] The second objective of this invention is to provide a Pseudomonas aeruginosa YHDXY-01 preparation for degrading creatinine and its application. The Pseudomonas aeruginosa YHDXY-01 preparation is made from the above-mentioned Pseudomonas aeruginosa YHDXY-01 strain used for biodegrading creatinine and can efficiently biodegrade creatinine.
[0007] Therefore, the first aspect of the present invention provides a strain of Pseudomonas asiatica YHDXY-01 that degrades creatinine, which can produce an enzyme that catalyzes the degradation of creatinine, and its accession number is CGMCCNo.37024.
[0008] The second aspect of the present invention provides a creatinine-degrading Pseudomonas aeruginosa YHDXY-01 preparation containing bacterial cells and / or crude enzymes of the Pseudomonas aeruginosa YHDXY-01 strain as described in the first aspect of the present invention.
[0009] According to some embodiments of the present invention, the Asian Pseudomonas aeruginosa YHDXY-01 preparation that degrades creatinine is a liquid preparation.
[0010] Preferably, in the liquid formulation for degrading creatinine, the cell concentration of Pseudomonas Asiana YHDXY-01 strain is (2-5) × 10⁻⁶. 8 / mL.
[0011] Preferably, in the liquid formulation for degrading creatinine, the protein concentration of the crude enzyme from Pseudomonas Asiana YHDXY-01 strain is 3-10 mg / mL.
[0012] In some other embodiments of the present invention, the creatinine-degrading Pseudomonas Asianae YHDXY-01 formulation is a solid powder formulation.
[0013] Preferably, in the solid powder formulation for degrading creatinine, the cell concentration of Pseudomonas Asiana YHDXY-01 strain is (1-6) × 10⁻⁶. 8 / g, more preferably (2-5)×10 8 / g.
[0014] Preferably, in the solid powder formulation for degrading creatinine, the crude enzyme protein content of Pseudomonas Asiana YHDXY-01 strain is 1-10 mg / g, more preferably 5-10 mg / g.
[0015] A third aspect of the present invention provides a method for preparing a creatinine-degrading Pseudomonas Asianae YHDXY-01 formulation as described in the second aspect of the present invention, comprising:
[0016] Step B: Inoculate the fermentation strain into the fermentation medium for fermentation culture to obtain the fermentation culture of Pseudomonas Asiana YHDXY-01 strain;
[0017] Step C: Centrifuge the fermentation culture of Pseudomonas Asianae YHDXY-01 strain to harvest the bacterial cells of Pseudomonas Asianae YHDXY-01 strain;
[0018] The fermentation strain was obtained from the corresponding Asian Pseudomonas strain YHDXY-01 through seed culture.
[0019] According to the present invention, the fermentation medium comprises, per 1L of water, the following components:
[0020] 5-10g of peptone; preferably 8-10g;
[0021] 5-10g of yeast powder; preferably 8-10g; and
[0022] 3-8 g of glucose; preferably 4-6 g;
[0023] Preferably, the pH value of the fermentation medium is 6-7;
[0024] More preferably, in step B, the fermentation culture temperature is 18-40℃, more preferably 35-38℃.
[0025] According to some embodiments of the present invention, the preparation method further includes:
[0026] Step K: The cell suspension of the Asian Pseudomonas YHDXY-01 strain is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of the Asian Pseudomonas YHDXY-01 strain.
[0027] Step L: Centrifuge the cell-free lysate of Pseudomonas Asianae YHDXY-01 strain and take the supernatant cell-free extract as the crude enzyme of Pseudomonas Asianae YHDXY-01 strain.
[0028] The low temperature is 0-4℃.
[0029] The fourth aspect of this invention provides the use of the *Pseudomonas aeruginosa* YHDXY-01 preparation described in the second aspect of this invention, or the *Pseudomonas aeruginosa* YHDXY-01 preparation prepared by the method described in the third aspect of this invention, in the preparation of creatinine-lowering agents, comprising:
[0030] Step D: Wash the cells of Pseudomonas Asianae YHDXY-01 strain with physiological saline to obtain pure cells of Pseudomonas Asianae YHDXY-01 strain.
[0031] Step E: In a physiological saline system, under low temperature conditions, the pure bacterial cells of Pseudomonas Asianum strain YHDXY-01 are broken by ultrasonication. After centrifugation, the supernatant is taken to obtain a cell-free extract as the crude enzyme pure product of Pseudomonas Asianum strain YHDXY-01.
[0032] Step F involves freeze-drying the purified bacterial cells and / or crude enzyme of Pseudomonas Asianum strain YHDXY-01, and then diluting the freeze-dried Pseudomonas Asianum strain YHDXY-01 to prepare a creatinine degradation agent.
[0033] The low temperature is 0-4℃.
[0034] In some embodiments of the present invention, in step F, the freeze-dried Pseudomonas Asianae YHDXY-01 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.
[0035] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Pseudomonas Asianis YHDXY-01 preparation to prepare a solid creatinine-lowering agent.
[0036] The inventors have discovered that the Pseudomonas Asianae strain YHDXY-01 and the enzymes it produces, which are used to biodegrade creatinine, are both capable of biodegrading creatinine and have significant application value and prospects in the prevention and treatment of kidney diseases such as high creatinine levels and their complications. Attached Figure Description
[0037] To facilitate understanding of the present invention, a more detailed description will be provided below with reference to the accompanying drawings, during which the above and other objects, features, and advantages of the present invention will become more apparent. The accompanying drawings are provided to further illustrate the present invention and form part of the specification. They are used together with the embodiments of the present invention to explain the invention and do not constitute a limitation thereof.
[0038] Figure 1 A molecular phylogenetic tree based on 16S rDNA is shown for screening Asian Pseudomonas YHDXY-01.
[0039] Figure 2 The kinetic curves of creatinine biodegradation by Pseudomonas aeruginosa YHDXY-01 are shown.
[0040] Figure 3 The kinetic curve of creatinine degradation catalyzed by the enzyme YHDXY-01 of Pseudomonas aeruginosa is shown.
[0041] strain preservation
[0042] The strain provided in this invention is classified and named *Pseudomonas asiatica*, isolated and identified by Beijing Yiran Biotechnology Co., Ltd., and deposited at the China General Microbiological Culture Collection Center (CGMCC; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on December 7, 2025, with accession number CGMCCNo. 37024. In this invention, this strain is named *Pseudomonas asiatica* strain YHDXY-01, also known as *Pseudomonas asiatica* YHDXY-01. Detailed Implementation
[0043] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.
[0044] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.
[0045] In this invention, the range of dosage concentration, temperature or other physical or chemical properties or characteristics, unless otherwise specified, covers or includes the upper and lower limits of that range.
[0046] I. Terminology
[0047] In this invention, the term "cell" refers to the live and / or dead cells of Pseudomonas aeruginosa.
[0048] In this invention, the term "crude enzyme" refers to the cell-free extract obtained by centrifuging and collecting the supernatant after the bacterial cells of Pseudomonas aeruginosa are broken.
[0049] The term "pure crude enzyme" used in this invention refers to a cell-free extract obtained by centrifuging and collecting the supernatant of pure Asian Pseudomonas cells after rupture.
[0050] The term "microbial preparation" as used in this invention refers to preparations of various forms made from microorganisms with medicinal value as raw materials, using traditional or modern biotechnology, and used for the prevention (health care), treatment, and diagnosis of various physiological symptoms in the human body.
[0051] The term "edible starch" as used in this invention refers to starch that meets the "National Standard for Edible Starch" (GB 31637-2016 National Food Safety Standard for Edible Starch).
[0052] The term "cell suspension" as used in this invention refers to the liquid obtained by adding approximately 50 mL of sterile physiological saline to a microbial fermentation culture after centrifugation to remove the supernatant, thereby suspending the precipitated microbial cells.
[0053] The "water" used in the invention for culture media or fermentation processes, unless otherwise specified, refers to sterile pure water obtained by filtration through a 0.22 µm filter membrane.
[0054] II. Implementation Plan
[0055] As mentioned earlier, existing dietary and pharmaceutical therapies for controlling creatinine levels in the human body are slow-acting, have limited efficacy, and suffer from side effects. Therefore, the inventors have conducted extensive and in-depth research on the biodegradation of creatinine.
[0056] The inventors noted that although lactic acid bacteria can promote gut health and reduce creatinine accumulation to some extent through a series of metabolic regulation, there are no reports of the strains used directly and efficiently biodegrading creatinine.
[0057] The inventors also noted that *Pseudomonas asiatica* is a bacterium. Although *Pseudomonas asiatica* has been applied in agriculture, environmental protection, and other fields, no research reports on its biodegradation of creatinine have been found to date.
[0058] Based on long-term research in microorganisms, the inventors have successfully screened a highly efficient pure strain of microorganism capable of biodegrading creatinine from the bacterial communities in the bottom sediment of industrial marine fish farming ponds. This strain and its produced enzymes are both highly efficient at biodegrading creatinine, possessing significant research value and promising application prospects in the efficient biodegradation and removal of creatinine. This invention is thus derived.
[0059] Therefore, the first aspect of the present invention provides a strain of Pseudomonas aeruginosa YHDXY-01 for biodegrading creatinine. Studies have found that the strain of Pseudomonas aeruginosa YHDXY-01 can produce one or more enzymes that can catalyze the degradation of creatinine.
[0060] The inventors first successfully screened a strain of *Pseudomonas asiatica*, YHDXY-01, from the bacterial community of bottom sediment in industrial marine fish farming ponds. Genomic DNA was extracted, and molecular identification, including PCR amplification and 16S rDNA sequencing, confirmed its identity as *Pseudomonas asiatica*. Based on the above, this strain was identified and named *Pseudomonas asiatica* strain YHDXY-01. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCCNo. 37024.
[0061] The inventors have discovered that fermentation culture of Pseudomonas Asianum strain YHDXY-01 produces bacterial cells containing one or more enzymes capable of catalyzing the degradation of creatinine. In this invention, the mixture of these enzymes is referred to as crude enzyme, or crude enzyme of Pseudomonas Asianum strain YHDXY-01.
[0062] Further research revealed that centrifugation of the cell-free lysate after bacterial cell disruption, and the collection of the supernatant cell-free extract, served as the crude enzyme for *Pseudomonas aeruginosa* strain YHDXY-01. This readily explains why both the cells and the crude enzyme of *Pseudomonas aeruginosa* strain YHDXY-01 can catalyze the degradation of creatinine.
[0063] The results showed that the Asian Pseudomonas strain YHDXY-01, with a cell concentration of 1.3 × 10⁻⁶, was effective in reducing bacterial counts from an initial concentration of 1.3 × 10⁻⁶. 8 / mL grew to 3.3×10 8 / mL, capable of degrading and removing 80.6% of creatinine at an initial concentration of 5 g / L within 48 hours.
[0064] The results also showed that the Asian Pseudomonas YHDXY-01 strain, with a crude enzyme protein concentration of 2.2 mg / mL, was able to completely degrade creatinine at an initial concentration of 500 mg / L within 24 hours.
[0065] Based on the above, the second to fourth aspects of the present invention further provide the use or application of the Asian Pseudomonas bacillus for biodegrading creatinine as described in the first aspect of the present invention.
[0066] Specifically, the second aspect of the present invention provides a biodegradable creatinine preparation of Pseudomonas Asiana YHDXY-01, which belongs to a biodegradable creatinine microbial preparation containing bacterial cells and / or crude enzymes of Pseudomonas Asiana YHDXY-01 strain as provided in the first aspect of the present invention.
[0067] In some preferred embodiments of the present invention, the Pseudomonas Asianae YHDXY-01 preparation contains bacterial cells of the Pseudomonas Asianae YHDXY-01 strain as described in the first aspect of the present invention.
[0068] According to some embodiments of the present invention, the biodegradable creatinine Pseudomonas Asiana YHDXY-01 preparation is a liquid preparation.
[0069] For example, in some embodiments of the present invention, the cell concentration of Pseudomonas aeruginosa strain YHDXY-01 in the liquid formulation of the biodegradable creatinine is (2-5) × 10⁻⁶. 8 / mL.
[0070] For example, in other embodiments of the invention, the crude enzyme protein concentration of Pseudomonas Asiana YHDXY-01 strain is 2-10 mg / mL in the liquid formulation of the biodegradable creatinine.
[0071] According to other embodiments of the present invention, the biodegradable creatinine Pseudomonas Asiana YHDXY-01 formulation is a solid powder formulation.
[0072] For example, in some embodiments of the present invention, the cell concentration of Pseudomonas Asiana YHDXY-01 strain in the biodegradable creatinine solid powder formulation is (1-6) × 10⁻⁶. 8 / g, preferably (2-5)×10 8 / g.
[0073] For example, in other embodiments of the present invention, the crude enzyme protein content of Pseudomonas Asiana YHDXY-01 strain in the solid powder formulation of the biodegradable creatinine is 1-10 mg / g, preferably 5-10 mg / g.
[0074] A third aspect of the present invention provides a method for preparing a biodegradable creatinine-containing Pseudomonas Asianae YHDXY-01 formulation as described in the second aspect of the present invention, comprising:
[0075] Step B: Inoculate the fermentation strain into the fermentation medium and ferment at 18-40℃, preferably 35-38℃, at a shaking speed of 100-300 rpm for 3-5 days to obtain the fermentation culture of Pseudomonas Asiana YHDXY-01 strain.
[0076] Step C: Centrifuge the fermentation culture of Pseudomonas Asianae YHDXY-01 strain to harvest the bacterial cells of Pseudomonas Asianae YHDXY-01 strain;
[0077] The fermentation strain was obtained from the corresponding Asian Pseudomonas strain YHDXY-01 through seed culture.
[0078] As is known to those skilled in the art, 16S rRNA is commonly used internationally for the molecular identification of prokaryotic microorganisms. Therefore, 16S rRNA can be used for comparison to obtain homology. Thus, the fermentation strain used in this invention is not limited to the field isolates used in this invention. 16S rDNA is the DNA sequence on the bacterial genome that encodes rRNA and exists in the genomes of all prokaryotic microorganisms. Figure 1 A molecular phylogenetic tree based on 16S rDNA of Pseudomonas Asianis strain YHDXY-01 is shown.
[0079] In step C above, the centrifugation process includes resuspending and washing the precipitate (i.e., the bacterial cells of Pseudomonas Asiana YHDXY-01 strain) obtained by centrifugation of the liquid fermentation culture with physiological saline, and then centrifuging it again to obtain the bacterial cells of Pseudomonas Asiana YHDXY-01 strain.
[0080] The present invention does not impose any particular restrictions on the centrifugal separation conditions in step C above. For example, in some cases, the analyte can be centrifuged for 10 min at 8000-10000 r / min.
[0081] According to the method of the present invention, the fermentation culture is a shaker or fermenter fermentation culture of the microbial strain, and the fermentation strain is inoculated into the fermentation medium in the form of a seed liquid. The inoculation amount of the seed liquid is 0.1%-1% (v / v), preferably 0.2%-0.5% (v / v), and more preferably 0.5% (v / v).
[0082] In some specific embodiments of the present invention, the above-mentioned fermentation culture medium, based on 1L of water, includes the following components in 1L of water:
[0083] 5-10g of peptone;
[0084] 5-10g of yeast powder; and
[0085] 3-8g of glucose.
[0086] Preferably, the above fermentation medium, based on 1L of water, includes the following components in 1L of water:
[0087] 8-10g of peptone;
[0088] 8-10g of yeast powder; and
[0089] 4-6 g of glucose.
[0090] More preferably, the initial pH of the fermentation medium is adjusted to 6-7 using a 40% (wt / v) sodium hydroxide solution and a 36% (v / v) hydrochloric acid solution.
[0091] According to some embodiments of the present invention, the preparation method of the Pseudomonas Asianae YHDXY-01 preparation of the present invention further includes step A, which involves seed culture before step B: picking a single colony of Pseudomonas Asianae YHDXY-01 provided by the present invention and inoculating it into 100 mL of fermentation liquid culture medium, and then culturing it in a shaker at 38°C and 200 r / min for 3 days to obtain the fermentation strain (seed liquid).
[0092] The inventors studied the effects of different temperatures on the growth of Pseudomonas Asianum YHDXY-01 and found that Pseudomonas Asianum YHDXY-01 grew rapidly at a temperature of 38℃.
[0093] According to some specific embodiments of the present invention, the preparation method further includes:
[0094] Step K: The cell suspension of the Asian Pseudomonas YHDXY-01 strain is subjected to cell disruption treatment in an ice-water bath (i.e., ice-water mixture, 0-4℃) to obtain cell-free lysate of the Asian Pseudomonas YHDXY-01 strain.
[0095] Step L: The cell-free lysate of Pseudomonas Asianae YHDXY-01 strain is centrifuged, and the supernatant cell-free extract is used as the crude enzyme of Pseudomonas Asianae YHDXY-01 strain.
[0096] The present invention does not impose any particular restrictions on the centrifugal separation conditions in step L above. For example, in some cases, the analyte can be centrifuged at 15,000-18,000 r / min for 10-20 min.
[0097] The fourth aspect of this invention provides the use of the biodegradable creatinine-producing Pseudomonas aeruginosa YHDXY-01 preparation described in the second aspect of this invention, or the biodegradable creatinine-producing Pseudomonas aeruginosa YHDXY-01 preparation prepared by the method described in the third aspect of this invention, in the preparation of creatinine-lowering agents, comprising:
[0098] Step D: Wash the cells of Pseudomonas Asianae YHDXY-01 strain with physiological saline to obtain pure cells of Pseudomonas Asianae YHDXY-01 strain.
[0099] Step E: In a physiological saline system, under low temperature conditions of 0-4℃, the pure bacterial cells of Pseudomonas Asianum strain YHDXY-01 are broken by ultrasonication. After centrifugation, the supernatant is taken to obtain a cell-free extract as the crude enzyme pure product of Pseudomonas Asianum strain YHDXY-01.
[0100] Step F involves freeze-drying the purified bacterial cells and / or crude enzymes of Pseudomonas Asianum strain YHDXY-01, and then diluting the freeze-dried Pseudomonas Asianum strain YHDXY-01 to prepare a drug for lowering creatinine.
[0101] In some embodiments of the present invention, in step F, the freeze-dried Pseudomonas Asianae YHDXY-01 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.
[0102] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Pseudomonas Asianis YHDXY-01 preparation to prepare a solid creatinine-lowering agent.
[0103] III. Related materials and testing methods in this invention
[0104] 1. Materials
[0105] The seawater sediment samples involved in this invention were obtained from an industrialized marine fish farm in Dalian, Liaoning Province.
[0106] 2. Detection Method
[0107] (1) The cell concentration in this invention is determined using the following method:
[0108] To determine the concentration of Pseudomonas Asianum YHDXY-01 cells, a culture of Pseudomonas Asianum YHDXY-01 was diluted with physiological saline and the cell concentration was directly measured using a flow cytometer (SYSMEX, Germany).
[0109] (2) The creatinine concentration in this invention is determined using the following method:
[0110] The creatinine in the liquid culture medium was fully dissolved in pure water according to the specified ratio. After dilution and centrifugation, an appropriate amount of supernatant was passed through a membrane, and the concentration of creatinine in the liquid culture medium was determined using a RID-20A high performance liquid chromatograph (Shimadzu).
[0111] (3) The crude enzyme protein concentration in this invention is determined using the following method:
[0112] Cell-free extract of Pseudomonas Asianae YHDXY-01 was diluted with phosphate buffer solution and then Coomassie Brilliant Blue G-250 dye reagent was added in proportion and reacted for 10 minutes. The absorbance was measured at 595 nm using a 722S visible spectrophotometer (Shanghai Lingguang), and the protein concentration was calculated using the standard curve method.
[0113] IV. Examples
[0114] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.
[0115] Example 1: Preparation of bacterial cells and crude enzymes of Pseudomonas aeruginosa strain YHDXY-01
[0116] (1) Prepare the growth medium for Pseudomonas Asianae YHDXY-01, which consists of (per liter): 10.0 g peptone, 10.0 g yeast extract, and 5.0 g glucose. Add 100 ml of the prepared liquid culture medium to a 500 ml Erlenmeyer flask, sterilize at high temperature and high pressure (121 ℃) for 20 minutes, and then sterilize again under ultraviolet irradiation in a clean workbench for 20 minutes.
[0117] (2) Under sterile conditions in a clean workbench, 0.5 ml of Pseudomonas Asiana YHDXY-01 bacterial suspension was inoculated into a triangular flask of liquid culture medium. After batch culture for 3 days at a temperature of 38 ℃ and a shaking speed of 200 r / min, Pseudomonas Asiana YHDXY-01 cells were harvested by centrifugation (8000 r / min, 10 min) and discarding the supernatant.
[0118] Figure 1 The strain we screened was most closely related to Pseudomonas aeruginosa, so it was named Pseudomonas aeruginosa YHDXY-01.
[0119] (3) Take 20 mL of Pseudomonas aeruginosa YHDXY-01 cell suspension and add it to a 50 mL glass tube. Then, insert the tube into ice water and use an ultrasonic cell disruptor to disrupt the Pseudomonas aeruginosa YHDXY-01 cells. The conditions are: ultrasonic power 400W, interval 2 s, ultrasonic oscillation 10 s, disruption time 15 min (5 minutes each time). After the cells are disrupted, centrifuge the cell disruption solution at 15000 rpm for 20 minutes, and then slowly pour out the supernatant as the cell-free extract (crude enzyme) of Pseudomonas aeruginosa YHDXY-01.
[0120] Example 2: Degradation of creatinine by Pseudomonas aeruginosa strain YHDXY-01 cells and crude enzymes
[0121] Based on different creatinine concentrations, cultured Pseudomonas Asianae YHDXY-01 cells and crude enzymes were added in a specific ratio as a rapid, safe, and efficient biocatalyst to achieve rapid and efficient degradation and removal of creatinine. The results are as follows: Figure 2 and Figure 3 As shown.
[0122] Figure 2This indicates that within 48 hours, the concentration of Pseudomonas Asianae YHDXY-01 cells decreased from 1.3 × 10⁻⁶. 8 / mL grew to 3.3×10 8 The concentration of creatinine was reduced by 80.6% to a concentration of 5 g / L, indicating that Pseudomonas Asianum YHDXY-01 has a strong biodegradation ability for creatinine.
[0123] Figure 3 The results indicate that the cell-free extract (crude enzyme) of Pseudomonas Asiana YHDXY-01 can catalyze the degradation of creatinine at a faster rate. At a protein concentration of 2.2 mg / mL, it can completely degrade creatinine with an initial concentration of 500 mg / L in 24 hours, demonstrating a very high creatinine degradation rate.
[0124] It should be noted that the embodiments described above are merely preferred embodiments of the present invention, used to explain the present invention, and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.
Claims
1. A strain of Pseudomonas asiatica YHDXY-01 that degrades creatinine, which produces an enzyme that catalyzes the degradation of creatinine, has the accession number CGMCCNo.37024.
2. A creatinine-degrading Pseudomonas aeruginosa YHDXY-01 preparation, comprising bacterial cells and / or crude enzymes of the Pseudomonas aeruginosa YHDXY-01 strain as described in claim 1.
3. The Pseudomonas Asianae YHDXY-01 preparation according to claim 2, characterized in that, The creatinine-degrading Pseudomonas Asianae YHDXY-01 preparation is a liquid preparation; preferably, in the creatinine-degrading liquid preparation, the cell concentration of Pseudomonas Asianae YHDXY-01 strain is (2-5) × 10⁻⁶. 8 / mL; and / or, in the liquid formulation for degrading creatinine, the protein concentration of the crude enzyme of Pseudomonas Asianae YHDXY-01 strain is 2-10 mg / mL.
4. The Pseudomonas Asianae YHDXY-01 preparation according to claim 2, characterized in that, The creatinine-degrading Pseudomonas Asianae YHDXY-01 preparation is a solid powder formulation; preferably, in the creatinine-degrading solid powder formulation, the cell concentration of Pseudomonas Asianae YHDXY-01 strain is (1-6) × 10⁻⁶. 8 g, more preferably (2-5)×10 8 / g; and / or, in the solid powder formulation for degrading creatinine, the protein content of the crude enzyme of Pseudomonas Asianae YHDXY-01 strain is 1-10 mg / g, more preferably 5-10 mg / g.
5. A method for preparing a creatinine-degrading Pseudomonas Asianae YHDXY-01 formulation as described in any one of claims 2-4, comprising: Step B: Inoculate the fermentation strain into the culture medium for fermentation culture to obtain the fermentation culture of Pseudomonas Asiana YHDXY-01 strain; Step C: Centrifuge the fermentation culture of Pseudomonas Asianae YHDXY-01 strain to harvest the bacterial cells of Pseudomonas Asianae YHDXY-01 strain; The fermentation strain was obtained from the corresponding Asian Pseudomonas strain YHDXY-01 through seed culture.
6. The preparation method according to claim 5, characterized in that, The fermentation medium, calculated per liter of water, comprises the following components per liter of water: 5-10g of peptone; preferably 8-10g; 5-10g of yeast powder; preferably 8-10g; as well as 3-8 g of glucose; preferably 4-6 g; Preferably, the pH value of the fermentation medium is 6-7; More preferably, in step B, the fermentation culture temperature is 18-40℃, more preferably 35-38℃.
7. The preparation method according to claim 5 or 6, characterized in that, The preparation method further includes: Step K: The cell suspension of the Asian Pseudomonas YHDXY-01 strain is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of the Asian Pseudomonas YHDXY-01 strain. Step L: Centrifuge the cell-free lysate of Pseudomonas Asianae YHDXY-01 strain and take the supernatant cell-free extract as the crude enzyme of Pseudomonas Asianae YHDXY-01 strain. The low temperature is 0-4℃.
8. The use of a creatinine-degrading Pseudomonas aeruginosa YHDXY-01 preparation as described in any one of claims 2-4, or a Pseudomonas aeruginosa YHDXY-01 preparation prepared by any one of claims 5-7, in the preparation of a creatinine-lowering agent, comprising: Step D: Wash the cells of Pseudomonas Asianae YHDXY-01 strain with physiological saline to obtain pure cells of Pseudomonas Asianae YHDXY-01 strain. Step E: In a physiological saline system, under low temperature conditions, the pure bacterial cells of Pseudomonas Asianum strain YHDXY-01 are broken by ultrasonication. After centrifugation, the supernatant is taken to obtain a cell-free extract as the crude enzyme pure product of Pseudomonas Asianum strain YHDXY-01. Step F involves freeze-drying the purified bacterial cells and / or crude enzyme of Pseudomonas Asianum strain YHDXY-01, and then diluting the freeze-dried Pseudomonas Asianum strain YHDXY-01 to prepare a creatinine degradation agent. The low temperature is 0-4℃.
9. The application according to claim 8, characterized in that, In step F, the freeze-dried Pseudomonas Asianae YHDXY-01 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.
10. The application according to claim 8, characterized in that, In step F, the freeze-dried Pseudomonas Asianae YHDXY-01 preparation is diluted with edible starch to prepare a solid creatinine-lowering agent.