CN118931741A - Preparation of Kluyveromyces marxianus YZC-01 for degrading uric acid and its application - Google Patents
Preparation of Kluyveromyces marxianus YZC-01 for degrading uric acid and its application Download PDFInfo
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- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A61P19/00—Drugs for skeletal disorders
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
- C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
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- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
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Abstract
本发明属于生物技术领域，涉及一种降解尿酸的马克斯克鲁维酵母YZC‑01株，其能够产生催化降解尿酸的酶。本发明还涉及一种降解尿酸的马克斯克鲁维酵母YZC‑01制剂，该马克斯克鲁维酵母YZC‑01制剂含有上述马克斯克鲁维酵母YZC‑01株的菌细胞和/或粗酶。研究结果表明，本发明所提供的用于生物降解尿酸的马克斯克鲁维酵母YZC‑01株及其所产生的酶对人体安全，且均能够高效生物降解尿酸，在去除尿酸用于防治痛风及相应并发症方面具有重要的应用前景。
The present invention belongs to the field of biotechnology, and relates to a strain of Kluyveromyces marxianus YZC-01 for degrading uric acid, which can produce an enzyme that catalyzes the degradation of uric acid. The present invention also relates to a preparation of Kluyveromyces marxianus YZC-01 for degrading uric acid, which contains bacterial cells and/or crude enzymes of the above-mentioned Kluyveromyces marxianus YZC-01 strain. The results of the study show that the Kluyveromyces marxianus YZC-01 strain for biodegrading uric acid provided by the present invention and the enzyme produced by it are safe for the human body, and can efficiently biodegrade uric acid, and have important application prospects in removing uric acid for the prevention and treatment of gout and corresponding complications.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid, and a preparation method and application thereof.
Background
In recent years, gout patients in China are rapidly increased due to the increase of the content of blood uric acid (Serum uric acid), and the gout patients form a serious threat to the health of people. Uric acid (Uric acid) is a final product of purine metabolism in humans, has a molecular formula of C 5H4N4O3, a molecular weight of 168, is slightly soluble in water, and is easily formed into crystals. About 80% of uric acid in humans is produced by endogenous small molecule compound synthesis and purine nucleotide catabolism, and the remaining 20% of uric acid is produced by degradation of exogenous purine ingested in the food.
Under normal conditions, uric acid production and excretion in the human body are maintained in a substantially dynamic balance, but uric acid is not degraded into more soluble allantoin due to lack of urate oxidase, thus preventing uric acid excretion through both the kidney and the intestinal tract. Uric acid production and/or excretion will result in elevated blood uric acid levels in humans, and thus hyperuricemia, with diagnostic criteria: fasting blood uric acid levels were above the threshold (male > 420. Mu. Mol/L, female > 360. Mu. Mol/L) 2 times a day. Uric acid is a final product of purine metabolism and can be degraded by urate oxidase into water-soluble allantoin in non-primate animals and is easily discharged, whereas human beings and primates lack urate oxidase to make uric acid not easily decomposed, so that the blood uric acid level is 3-10 times higher than that of non-primate animals, which is also a main cause of human beings suffering from hyperuricemia and gout.
The current treatments or improvements of hyperuricemia are mainly divided into drug treatment and dietary intervention, wherein drug treatment is the most common way to control hyperuricemia, and common drugs include allopurinol, febuxostat, phenylbromarone, urate oxidase (labyrine, prim) and the like, and the blood uric acid level is controlled mainly by inhibiting the activity of xanthine oxidase to reduce the generation of uric acid or reducing the reabsorption of uric acid to increase the excretion of uric acid. The main mechanism of action of urate oxidase is to further degrade uric acid into more soluble allantoin, thus facilitating excretion from the kidneys. Although chemical drugs can play a certain role in controlling hyperuricemia, a series of side effects such as anaphylaxis, liver injury, renal dysfunction and the like are not ignored. Dietary intervention is mainly aimed at alleviating hyperuricemia by limiting the intake of high purine foods, beer and fructose, and although blood uric acid levels can be controlled to some extent, the effect is not ideal. Therefore, the method for safely and efficiently controlling hyperuricemia is researched and becomes a key scientific research difficult problem to be solved urgently in the field of biological medicine at present.
Disclosure of Invention
Aiming at the problems of insignificant effect and side effects of medicines or foods for degrading and removing uric acid in the prior art, the invention provides a Kluyveromyces marxianus YZC-01 strain for degrading uric acid, bacterial cells of the strain and enzymes produced by the strain are safe to human bodies, can efficiently biodegrade uric acid, and has important application prospects in removing uric acid.
The invention also aims to provide a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid and application thereof, wherein the Kluyveromyces marxianus YZC-01 preparation is prepared from the Kluyveromyces marxianus YZC-01 strain for biodegrading uric acid, and can biodegrade uric acid efficiently.
To this end, the first aspect of the present invention provides a Kluyveromyces marxianus (Kluyveromyces marxianus) YZC-01 strain for degrading uric acid, which is capable of producing an enzyme for catalyzing the degradation of uric acid, and the preservation number of the enzyme is CGMCCNo.29514.
In some embodiments of the invention, the Kluyveromyces marxianus YZC-01 strain can degrade and remove uric acid with the initial concentration of 2g/L in 72 hours at the initial concentration of 2X 107/mL of bacterial cells.
In other embodiments of the present invention, crude enzyme produced by Kluyveromyces marxianus YZC-01 strain is capable of completely removing uric acid having an initial concentration of 2g/L in 24 hours at a protein concentration of 8.0 mg/mL.
The second aspect of the invention provides a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid, which contains bacterial cells and/or crude enzyme of the Kluyveromyces marxianus YZC-01 strain; preferably, the Kluyveromyces marxianus YZC-01 preparation contains the Kluyveromyces marxianus YZC-01 strain according to the first aspect of the invention.
In some embodiments of the invention, the uric acid degrading Kluyveromyces marxianus YZC-01 formulation is a liquid formulation; preferably, in the uric acid degrading liquid preparation, the concentration of bacterial cells of Kluyveromyces marxianus YZC-01 strain is (2-5). Times.107/mL; and/or in the uric acid degrading liquid preparation, the protein concentration of crude enzyme of Kluyveromyces marxianus YZC-01 strain is 5-10mg/mL.
In other embodiments of the present invention, the uric acid degrading Kluyveromyces marxianus YZC-01 formulation is a solid powder formulation; preferably, in the uric acid degrading solid powder preparation, the content of the bacterial cells of Kluyveromyces marxianus YZC-01 strain is (1-6). Times.107/g, more preferably (2-5). Times.107/g; and/or, in the uric acid degrading solid powder preparation, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 5-10mg/g, more preferably 8-10mg/g.
In a third aspect, the invention provides a preparation method of a uric acid degrading Kluyveromyces marxianus YZC-01 preparation, which comprises the following steps:
Step B, inoculating a fermentation strain into a fermentation medium for fermentation culture to obtain a fermentation culture of Kluyveromyces marxianus YZC-01 strain;
Step C, performing centrifugal separation treatment on a fermentation culture of the Kluyveromyces marxianus YZC-01 strain, and obtaining bacterial cells of the Kluyveromyces marxianus YZC-01 strain;
Wherein, the fermentation strain is obtained by seed culture of the corresponding Kluyveromyces marxianus YZC-01 strain. According to the invention, the fermentation medium comprises the following components in 1L of water, calculated as 1L of water:
5-10g of peptone; preferably 8-10g;
5-10g of yeast powder; preferably 8-10g; and
Glucose 3-8g; preferably 4-6g;
preferably, the pH of the fermentation medium is 7-8;
further preferably, in step B, the temperature of the fermentation culture is 18-40 ℃, preferably 30-35 ℃.
According to some embodiments of the invention, the method further comprises:
step K, performing cell disruption treatment on the cell suspension of the Kluyveromyces marxianus YZC-01 strain at a low temperature to obtain a cell-free disruption solution of the Kluyveromyces marxianus YZC-01 strain;
Step L, performing centrifugal separation on a cell-free crushing liquid of the Kluyveromyces marxianus YZC-01 strain, and taking a supernatant cell-free extracting liquid as crude enzyme of the Kluyveromyces marxianus YZC-01 strain;
Wherein the low temperature is 0-4 ℃.
The fourth aspect of the invention provides an application of the uric acid degrading Kluyveromyces marxianus YZC-01 preparation in preparing uric acid degrading medicaments, which comprises the following steps:
Step D, washing bacterial cells of the Kluyveromyces marxianus YZC-01 strain by using physiological saline to obtain a bacterial cell pure product of the Kluyveromyces marxianus YZC-01 strain;
E, crushing a bacterial cell pure product of the Kluyveromyces marxianus YZC-01 strain by using ultrasonic waves in a physiological saline system at a low temperature, centrifuging, and taking supernatant to obtain a cell-free extract serving as a crude enzyme pure product of the Kluyveromyces marxianus YZC-01 strain;
step F, freeze-drying a bacterial cell pure product and/or a crude enzyme pure product of the Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare a medicament for degrading uric acid;
Wherein the low temperature is 0-4 ℃.
In some embodiments of the present invention, in step F, the lyophilized Kluyveromyces marxianus YZC-01 formulation is diluted with physiological saline to prepare a liquid uric acid lowering agent.
In other embodiments of the present invention, in step F, the lyophilized Kluyveromyces marxianus YZC-01 formulation is diluted with an edible starch to form a solid uric acid lowering agent.
In some preferred embodiments of the invention, the uric acid lowering agent is an oral formulation.
Researches show that the Kluyveromyces marxianus YZC-01 strain for biodegrading uric acid and the enzyme produced by the strain are safe to human bodies, can biodegrade uric acid, and have important application prospects in the aspect of efficiently removing uric acid for treating hyperuricemia and corresponding complications of human bodies.
Drawings
In order that the invention may be readily understood, the invention will be described with reference to the accompanying drawings.
FIG. 1 shows a molecular evolution tree based on 18S rDNA for screening Kluyveromyces marxianus YZC-01.
FIG. 2 is a kinetic profile of Kluyveromyces marxianus YZC-01 for biodegradation of uric acid.
FIG. 3 is a kinetic profile of Kluyveromyces marxianus YZC-01 enzyme-catalyzed degradation of uric acid.
Preservation of bacterial species
Kluyveromyces marxianus (Kluyveromyces marxianus), isolated and identified by Beijing Corp, yi Xie Bio-technology Co., ltd.) has been deposited at China general microbiological culture Collection center (CGMCC; address: china center for general purpose, academy of sciences of China, no. 3, north Star, chaoyang, beijing) with a date of deposition: 2024 1 month 4, deposit number: CGMCC No.29514. The strain is named as Kluyveromyces marxianus YZC-01 strain (Kluyveromyces marxianus strain YZC-01) in the invention, and is also named as Kluyveromyces marxianus YZC-01.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I, terminology
The term "cell" as used herein refers to a live cell and/or dead cell of Kluyveromyces marxianus.
The term "crude enzyme" as used herein refers to a cell-free extract obtained by disrupting cells of Kluyveromyces marxianus and then centrifuging to obtain a supernatant.
The term "crude enzyme purified product" as used herein refers to a cell-free extract obtained by crushing a pure bacterial cell product of Kluyveromyces marxianus and then centrifuging to obtain a supernatant, relative to crude enzyme.
The term "microbial preparation" in the present invention refers to a preparation which is produced by using a microorganism having medical research value as a raw material and is used for preventing (health care), treating and diagnosing various physiological symptoms of human body by using a traditional technology or a modern biotechnology.
The term "edible starch" as used herein refers to starch which meets the national standard of edible starch (GB 31637-2016 national standard of food safety edible starch).
The term "water" used in the culture medium or the fermentation culture process of the invention refers to sterile pure water obtained by filtration through a 0.22. Mu. Filter, unless otherwise specified.
II, embodiment
As previously mentioned, existing dietetic and pharmaceutical therapies for the control of uric acid in humans have slow and insignificant efficacy and side effects. Accordingly, the present inventors have conducted extensive and intensive studies on the biodegradation of uric acid.
The inventors noted that although lactic acid bacteria promote intestinal health and reduce uric acid accumulation to some extent through a series of metabolic regulation, the strain used has not been reported to biodegrade uric acid directly and efficiently.
In recent years, research on human microorganisms is a research hotspot and a front edge of life science, and although domestic and foreign scholars screen microorganism strains capable of inhibiting uric acid synthesis and degrading uric acid precursors inosine, guanosine and the like from traditional foods, no evidence that kluyveromyces marxianus can directly and efficiently biodegrade uric acid is found.
The inventor also notes that Kluyveromyces marxianus (Kluyveromycesmarxianus) belongs to eukaryotic microorganisms and is yeast which can be directly eaten by people who pass Wei Jian commission approval in China. Although kluyveromyces marxianus can promote human intestinal health and improve human immune function, has been widely applied to the fields of food, fermentation, beer and beverage production and the like, research reports on biodegradation of uric acid have not been found yet.
Based on the long-term study of microorganisms, the inventor successfully screens a Kluyveromyces marxianus YZC-01 (Kluyveromyces marxianus YZC-01) from intestinal bacteria of healthy human bodies, and discovers that cells of the strain and generated enzymes can degrade uric acid with initial concentration of 2g/L within 72h and 24h respectively through research and exploration, so that the method has very important research value and important application prospect in the aspect of removing uric acid through high-efficiency biodegradation. The present invention has been achieved thereby.
Thus, kluyveromyces marxianus YZC-01 strain for biodegradation of uric acid, which is capable of producing an enzyme catalyzing degradation of uric acid, is related to the first aspect of the present invention.
The inventor firstly successfully screens a Kluyveromyces marxianus YZC-01 strain from intestinal flora of a healthy human body. The genomic DNA was extracted, and the resulting mixture was amplified by PCR and identified as Kluyveromyces marxianus (Kluyveromyces marxianus) by molecular analysis based on 18S rDNA sequencing, and the strain was identified and designated as Kluyveromyces marxianus YZC-01 strain (Kluyveromyces marxianusstrain YZC-01) based on the above. The strain is preserved in China general microbiological culture Collection center, with the preservation number: cgmccno.29514.
The present inventors have found that Kluyveromyces marxianus YZC-01 is fermented and cultured, and that the yeast cells produced during the fermentation and culture process contain one or more enzymes capable of catalyzing and degrading uric acid, and that the mixture of these enzymes is referred to as crude enzyme or Kluyveromyces marxianus YZC-01 crude enzyme in the present invention.
Further research shows that the cell-free disrupted solution after yeast cell disruption is subjected to centrifugal separation, and the supernatant cell-free extract is taken as a crude enzyme of Kluyveromyces marxianus YZC-01. From this, it is easily understood that both the cells of Kluyveromyces marxianus YZC-01 strain and the crude enzyme are capable of catalyzing the degradation of uric acid.
The research result shows that the Kluyveromyces marxianus YZC-01 strain can completely degrade and remove uric acid with the initial concentration of 2g/L in 72 hours under the condition that the initial concentration of bacterial cells is 2X 10 7/mL.
The crude enzyme produced by the Kluyveromyces marxianus YZC-01 strain can degrade uric acid with the initial concentration of 2g/L in 24 hours under the condition of the protein concentration of 8.0 mg/mL.
Based on the above, the second to fourth aspects of the present invention further provide the use or application of kluyveromyces marxianus for biodegradation of uric acid according to the first aspect of the present invention.
Specifically, the second aspect of the invention provides a Kluyveromyces marxianus YZC-01 preparation for biodegradation of uric acid, which belongs to a microbial preparation for biodegradation of uric acid, and contains bacterial cells and/or crude enzymes of the Kluyveromyces marxianus YZC-01 strain.
In some preferred embodiments of the present invention, the Kluyveromyces marxianus YZC-01 preparation contains bacterial cells of the Kluyveromyces marxianus YZC-01 strain according to the first aspect of the present invention.
According to some embodiments of the invention, the uric acid-biodegradable Kluyveromyces marxianus YZC-01 formulation is a liquid formulation.
In some embodiments of the invention, the concentration of the bacterial cells of Kluyveromyces marxianus YZC-01 strain in the liquid preparation for biodegradation of uric acid is (2-5). Times.10 7/mL.
In other embodiments of the present invention, the protein concentration of the crude enzyme of Kluyveromyces marxianus YZC-01 strain in the liquid preparation for biodegradation of uric acid is 5-10mg/mL.
According to other embodiments of the present invention, the Kluyveromyces marxianus YZC-01 formulation for biodegradation of uric acid is a solid powder formulation.
In some embodiments of the present invention, the Kluyveromyces marxianus YZC-01 strain has a bacterial cell content of (1-6). Times.10 7/g, preferably (2-5). Times.10 7/g in the solid powder preparation for biodegradation of uric acid.
In other embodiments of the invention, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain in the solid powder preparation for biodegradation of uric acid is 5-10mg/g, preferably 8-10mg/g.
In a third aspect, the invention provides a preparation method of Kluyveromyces marxianus YZC-01 preparation for biodegradation of uric acid, which comprises the following steps:
step B, inoculating fermentation strains into a fermentation medium, and carrying out fermentation culture for 3-5 days at the temperature of 18-40 ℃, preferably 30-35 ℃ and the rotation speed of a shaking table of 100-300 rpm to obtain a fermentation culture of Kluyveromyces marxianus YZC-01 strain;
Step C, performing centrifugal separation treatment on a fermentation culture of the Kluyveromyces marxianus YZC-01 strain, and obtaining bacterial cells of the Kluyveromyces marxianus YZC-01 strain;
wherein, the fermentation strain is obtained by seed culture of the corresponding Kluyveromyces marxianus YZC-01 strain.
As known to those skilled in the art, at present, 18S rRNA is commonly used internationally for molecular identification of eukaryotic microorganisms, and thus 18S rRNA can be used for comparison of similarity to obtain homology. The fermentation strain used in the present invention is not limited to the field isolate used in the present invention, and 18S rDNA is a DNA sequence corresponding to the encoded rRNA on the bacterial chromosome, and is present in the genome of all eukaryotic microorganisms. FIG. 1 shows a molecular evolution tree of Kluyveromyces marxianus YZC-01 strain based on 18S rDNA.
In the step C, the centrifugal separation treatment comprises the steps of carrying out centrifugal separation treatment on the sediment (namely the bacterial cells of the Kluyveromyces marxianus YZC-01 strain) obtained by the centrifugal separation treatment on the liquid fermentation culture, carrying out resuspension washing by using physiological saline, and then carrying out centrifugal separation treatment to obtain the bacterial cells of the Kluyveromyces marxianus YZC-01 strain.
The conditions for the centrifugation in the above step C are not particularly limited in the present invention, and in some embodiments of the present invention, for example, the to-be-separated substance may be centrifuged at 8000-10000r/min for 10min.
According to the method of the invention, the fermentation culture is a shaking table or a fermentation tank for fermentation culture of strains, and the fermentation strains are inoculated into a fermentation culture medium in the form of seed liquid. The inoculation amount of the seed liquid is 0.1% -1% (v/v); preferably, the seed solution is inoculated in an amount of 0.2% -0.5% (v/v); further preferably, the seed solution is inoculated in an amount of 0.5% (v/v).
Specifically, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
5-10g of peptone;
5-10g of yeast powder; and
Glucose 3-8g.
Preferably, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
8-10g of peptone;
8-10g of yeast powder; and
Glucose 4-6g.
In some embodiments of the invention, 40% (wt/v) sodium hydroxide solution and 36% (v/v) hydrochloric acid solution are used to adjust the initial pH of the fermentation medium to 6-8.
According to some embodiments of the present invention, the method for preparing kluyveromyces marxianus YZC-01 formulation according to the present invention further comprises a step a of seed culture before the step B: the Kluyveromyces marxianus YZC-01 monoclonal colony provided by the invention is picked and inoculated into 100mL fermentation liquid culture medium, and the fermentation strain (seed liquid) is prepared after shaking culture for 3 days at the temperature of 30 ℃ and the rotating speed of 200 r/min.
The inventors studied the effect of different temperatures on the growth of Kluyveromyces marxianus YZC-01 and found that Kluyveromyces marxianus YZC-01 grows fast at a temperature of 30 ℃.
According to some embodiments of the invention, the method further comprises:
step K, performing cell disruption treatment on the cell suspension of the Kluyveromyces marxianus YZC-01 strain in ice water bath (namely ice water mixture, 0-4 ℃) to obtain a cell-free disruption solution of the Kluyveromyces marxianus YZC-01 strain;
and step L, carrying out centrifugal separation on the acellular disruption solution of the Kluyveromyces marxianus YZC-01 strain, and taking the supernatant acellular extraction solution as crude enzyme of the Kluyveromyces marxianus YZC-01 strain.
The conditions for the centrifugation in the above step L are not particularly limited in the present invention, and in some embodiments of the present invention, for example, the to-be-separated substance may be centrifuged at 15000-18000r/min for 10-20min.
The fourth aspect of the invention provides an application of the Kluyveromyces marxianus YZC-01 preparation for biodegradation of uric acid in the second aspect of the invention or the Kluyveromyces marxianus YZC-01 preparation for biodegradation of uric acid prepared by the preparation method in the third aspect of the invention in preparation of uric acid-reducing medicaments, which comprises the following steps:
Step D, washing bacterial cells of the Kluyveromyces marxianus YZC-01 strain by using physiological saline to obtain a bacterial cell pure product of the Kluyveromyces marxianus YZC-01 strain;
E, in a physiological saline system, under the low temperature condition of 0-4 ℃, crushing a bacterial cell pure product of the Kluyveromyces marxianus YZC-01 strain by adopting ultrasonic waves, and taking a supernatant after centrifugal treatment to obtain a cell-free extract as a crude enzyme pure product of the Kluyveromyces marxianus YZC-01 strain;
And F, freeze-drying the pure bacterial cell product and/or crude enzyme product of the Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare the uric acid reducing medicament.
In some embodiments of the present invention, in step F, the lyophilized Kluyveromyces marxianus YZC-01 formulation is diluted with physiological saline to prepare a liquid uric acid lowering agent.
In other embodiments of the present invention, in step F, the lyophilized Kluyveromyces marxianus YZC-01 formulation is diluted with an edible starch to form a solid uric acid lowering agent.
In some preferred embodiments of the invention, the uric acid lowering agent is an oral formulation.
III, related materials and detection methods in the present invention
1. Material
The intestinal tract fecal bacteria capsule sample of the healthy human body is purchased from Beijing Fuma Tex biotechnology Co., ltd, and is licensed for research and development activities.
2. Detection method
(1) The cell concentration in the invention is determined by the following method:
Measurement of the cell concentration of Kluyveromyces marxianus YZC-01, taking a Kluyveromyces marxianus YZC-01 culture, diluting by a certain multiple with physiological saline, and directly measuring the cell concentration by a flow cytometer (SYSMEX in Germany).
(2) The uric acid concentration in the invention is measured by the following method:
The uric acid concentration is measured by a high performance liquid chromatograph (Shimadzu LC-20 AT), specifically a chromatographic column: kromasil C18 (4.6x250mm, 5 μ particle size); the mobile phase is methanol: 0.5% aqueous acetic acid (10:90); the detection wavelength is 283nm respectively; flow rate: 1mL/min; sample injection amount: 20. Mu.L; the temperature was 35 ℃.
(3) The concentration of crude enzyme protein in the invention is determined by the following method:
Taking a cell-free extract of Kluyveromyces marxianus YZC-01, diluting by a certain multiple by a phosphate buffer solution, adding a coomassie brilliant blue G-250 dye reagent proportionally, reacting for 10 minutes, measuring absorbance at 595nm by using a 722S visible spectrophotometer (Shanghai prismatic light), and calculating protein concentration by adopting a standard curve method.
III, examples
The present invention will be specifically described below by way of specific examples. The experimental methods described below, unless otherwise specified, are all laboratory routine methods. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
(1) Preparing a growth medium of Kluyveromyces marxianus YZC-01, wherein the growth medium comprises the following components (per liter): 10.0g of peptone, 10.0g of yeast powder and 5.0g of glucose. 100ml of the prepared liquid medium was added to a 500 ml Erlenmeyer flask, sterilized at high temperature and high pressure (121 ℃) for 20 minutes, and then sterilized under ultraviolet irradiation in a clean bench for 20 minutes.
(2) Inoculating 0.5ml of Kluyveromyces marxianus YZC-01 bacterial liquid in a triangular flask liquid culture medium under the aseptic condition in a clean workbench, carrying out batch culture for 3 days at the temperature of 30 ℃ and the rotation speed of a shaking table of 200 rpm, and obtaining the Kluyveromyces marxianus YZC-01 cells by a method of pouring out supernatant after centrifugation (8000 rpm, 10 minutes).
20ML of Kluyveromyces marxianus YZC-01 cell suspension is taken and added into a 50mL glass tube, and then the glass tube is inserted into ice water, and the Kluyveromyces marxianus YZC-01 cells are crushed by using an ultrasonic cell crusher, provided that: ultrasonic power 400W, interval 2 seconds, ultrasonic oscillation 10 seconds, crushing time 15 minutes (5 minutes each). After completion of cell disruption, the cell disruption solution was centrifuged at 15000 rpm for 20 minutes, and then the supernatant was slowly poured out as a cell-free extract (crude enzyme) of Kluyveromyces marxianus YZC-01.
(3) According to different concentrations of uric acid, kluyveromyces marxianus YZC-01 cells prepared by culture and crude enzyme are used as a quick, safe and efficient biocatalyst, and are added according to a certain proportion, so that the aim of quickly and efficiently degrading and removing uric acid is fulfilled.
FIG. 1 shows that the strain we screened was closest to Kluyveromyces marxianus and was therefore designated Kluyveromyces marxianus YZC-01 strain.
FIG. 2 shows that uric acid with an initial concentration of 2g/L can be completely degraded within 72 hours at an initial cell concentration of 2.0X10 7/mL of Kluyveromyces marxianus YZC-01, which shows that Kluyveromyces marxianus YZC-01 has a strong biodegradability to uric acid.
FIG. 3 shows that the cell-free extract (crude enzyme) of Kluyveromyces marxianus YZC-01 can catalyze and degrade uric acid at a faster rate, and uric acid with an initial concentration of 2g/L can be completely degraded in 24 hours at a protein concentration of 8.0mg/mL, so that the uric acid degradation rate is higher.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, not to be construed as limiting the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.
Claims (10)
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