Max Kluyveromyces marxianus YZC-01 Preparation for Degrading Uric Acid and Its Application

Abstract

The present invention belongs to the field of biotechnology and relates to Kluyveromyces marxianus strain YZC-01 for degrading uric acid, which can produce an enzyme for catalytically degrading uric acid. The present invention also relates to a Kluyveromyces marxianus strain YZC-01 preparation for degrading uric acid, and the Kluyveromyces marxianus strain YZC-01 preparation contains bacterial cells and / or crude enzymes of the above-mentioned Kluyveromyces marxianus strain YZC-01. Research results show that the Kluyveromyces marxianus strain YZC-01 for biodegradation of uric acid provided by the present invention and the enzyme produced by the strain are safe for the human body, and both can efficiently biodegrade uric acid, and have important application prospects in removing uric acid for preventing and treating gout and corresponding complications.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid, its preparation method and application. Background Art

[0002] In recent years, the number of gout patients caused by the increase in serum uric acid content in China has increased sharply, posing a serious threat to people's health. Uric acid is the end product of purine metabolism in the human body, and its molecular formula is C 5 H 4 N 4 O 3 , with a molecular weight of 168, slightly soluble in water and easy to form crystals. Approximately 80% of the uric acid in the human body is synthesized from endogenous small molecule compounds and produced by the catabolism of purine nucleotides, and the remaining 20% of the uric acid is generated by the degradation of exogenous purines ingested from food.

[0003] Under normal conditions, the generation and excretion of uric acid in the human body are basically in dynamic balance. However, due to the lack of uricase in humans, uric acid cannot be degraded into more soluble allantoin, thus hindering the excretion of uric acid through the kidneys and intestines. Excessive production and / or insufficient excretion of uric acid will lead to an increase in the serum uric acid level in the human body, resulting in hyperuricemia. The diagnostic criteria are: two fasting serum uric acid levels on different days are higher than the threshold (male > 420 μmol / L, female > 360 μmol / L). As the end product of purine metabolism, uric acid can be degraded into water-soluble allantoin and easily excreted by uricase in non-primate animals. However, humans and primates lack uricase, making uric acid not easily decomposed, so the serum uric acid level is 3-10 times higher than that of non-primate animals. This is also the main reason why humans are prone to hyperuricemia and gout.

[0004] At present, the treatment or improvement of hyperuricemia is mainly divided into drug therapy and dietary intervention. Drug therapy is the most common way to control hyperuricemia. Commonly used drugs include allopurinol, febuxostat, benzbromarone and urate oxidase (rasburicase, purikacil), etc., which mainly reduce the production of uric acid by inhibiting the activity of xanthine oxidase or increase the excretion of uric acid by reducing the reabsorption of uric acid, so as to control the level of blood uric acid. The main mechanism of action of urate oxidase is to further degrade uric acid into more soluble allantoin, so it is easily excreted from the kidneys. Although chemical drugs can play a certain role in controlling hyperuricemia, a series of side effects such as allergic reactions, liver damage, and abnormal renal function cannot be ignored. Dietary intervention mainly relieves hyperuricemia by limiting the intake of high-purine foods, beer and fructose. Although it can control the level of blood uric acid to a certain extent, the effect is not ideal. Therefore, studying safer and more efficient methods to control hyperuricemia has become a key scientific research problem that needs to be solved in the current biomedical field. Summary of the invention

[0005] One of the purposes of the present invention is to address the problem that the drugs or foods for degrading and removing uric acid in the prior art are not effective and have side effects, and to provide a Kluyveromyces marxianus YZC-01 strain for degrading uric acid. The bacterial cells of the strain and the enzymes produced by the strain are safe for the human body, and both can efficiently biodegrade uric acid, and have important application prospects in removing uric acid.

[0006] The second object of the present invention is to provide a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid and its application. The Kluyveromyces marxianus YZC-01 preparation is made from the above-mentioned Kluyveromyces marxianus YZC-01 strain for biodegrading uric acid and can efficiently biodegrade uric acid.

[0007] To this end, the first aspect of the present invention provides a Kluyveromyces marxianus YZC-01 strain for degrading uric acid, which is capable of producing an enzyme that catalyzes the degradation of uric acid, and its deposit number is CGMCC No.29514.

[0008] In some embodiments of the present invention, the Kluyveromyces marxianus YZC-01 strain can completely degrade and remove uric acid with an initial concentration of 2 g / L within 72 hours at an initial bacterial cell concentration of 2×10 7 / mL.

[0009] In other embodiments of the present invention, the crude enzyme produced by the Kluyveromyces marxianus strain YZC-01 can completely remove uric acid with an initial concentration of 2 g / L within 24 hours at a protein concentration of 8.0 mg / mL.

[0010] In a second aspect of the present invention, there is provided a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid, which contains the bacterial cells and / or crude enzyme of the Kluyveromyces marxianus YZC-01 strain as described in the first aspect of the present invention; preferably, the Kluyveromyces marxianus YZC-01 preparation contains the Kluyveromyces marxianus YZC-01 strain as described in the first aspect of the present invention.

[0011] In some embodiments of the present invention, the Kluyveromyces marxianus YZC-01 preparation for degrading uric acid is a liquid preparation; preferably, in the liquid preparation for degrading uric acid, the concentration of the bacterial cells of the Kluyveromyces marxianus YZC-01 strain is (2-5)×107 / mL; and / or, in the liquid preparation for degrading uric acid, the protein concentration of the crude enzyme of the Kluyveromyces marxianus YZC-01 strain is 5-10 mg / mL.

[0012] In some other embodiments of the present invention, the Kluyveromyces marxianus YZC-01 preparation for degrading uric acid is a solid powder preparation; preferably, in the solid powder preparation for degrading uric acid, the content of the bacterial cells of the Kluyveromyces marxianus YZC-01 strain is (1-6)×107 / g, more preferably (2-5)×107 / g; and / or, in the solid powder preparation for degrading uric acid, the protein content of the crude enzyme of the Kluyveromyces marxianus YZC-01 strain is 5-10 mg / g, more preferably 8-10 mg / g.

[0013] In a third aspect of the present invention, there is provided a method for preparing a Kluyveromyces marxianus YZC-01 preparation for degrading uric acid as described in the second aspect of the present invention, which includes:

[0014] Step B: inoculating the fermentation strain into a fermentation medium for fermentation culture to obtain a fermentation culture of the Kluyveromyces marxianus YZC-01 strain;

[0015] Step C: centrifuging and separating the fermentation culture of the Kluyveromyces marxianus YZC-01 strain to harvest the bacterial cells of the Kluyveromyces marxianus YZC-01 strain;

[0016] wherein, the fermentation strain is obtained by seed culture from the corresponding Kluyveromyces marxianus YZC-01 strain. According to the present invention, based on 1 L of water, the fermentation medium includes the following components in 1 L of water:

[0017] Peptone 5-10 g; preferably 8-10 g;

[0018] Yeast powder 5-10 g; preferably 8-10 g; and

[0019] Glucose 3-8 g; preferably 4-6 g;

[0020] Preferably, the pH value of the fermentation medium is 7-8;

[0021] More preferably, in step B, the temperature of the fermentation culture is 18-40 °C, preferably 30-35 °C.

[0022] According to some embodiments of the present invention, the preparation method further includes:

[0023] Step K, subjecting the cell suspension of Kluyveromyces marxianus YZC-01 strain to cell disruption treatment under low temperature conditions to obtain a cell-free lysate of Kluyveromyces marxianus YZC-01 strain;

[0024] Step L, centrifuging the cell-free lysate of Kluyveromyces marxianus YZC-01 strain, and taking the supernatant cell-free extract as the crude enzyme of Kluyveromyces marxianus YZC-01 strain;

[0025] Wherein, the temperature of the low temperature is 0-4 °C.

[0026] The fourth aspect of the present invention provides an application of the Kluyveromyces marxianus YZC-01 preparation for degrading uric acid according to the second aspect of the present invention or the Kluyveromyces marxianus YZC-01 preparation for degrading uric acid prepared by the preparation method according to the third aspect of the present invention in the preparation of anti-hyperuricemic agents, which includes:

[0027] Step D, washing the bacterial cells of Kluyveromyces marxianus YZC-01 strain with physiological saline to obtain pure bacterial cells of Kluyveromyces marxianus YZC-01 strain;

[0028] Step E, in a physiological saline system, under low temperature conditions, ultrasonically disrupting the pure bacterial cells of Kluyveromyces marxianus YZC-01 strain, and taking the supernatant after centrifugation to obtain a cell-free extract as the pure crude enzyme of Kluyveromyces marxianus YZC-01 strain;

[0029] Step F, freeze-drying the pure bacterial cells and / or pure crude enzyme of Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare an agent for degrading uric acid;

[0030] Wherein, the temperature of the low temperature is 0-4 °C.

[0031] In some embodiments of the present invention, in step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with physiological saline to prepare a liquid anti-hyperuricemic agent.

[0032] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Kluyveromyces marxianus YZC-01 preparation to make a solid uric acid-lowering medicament.

[0033] In some preferred embodiments of the present invention, the uric acid-lowering medicament is an oral preparation.

[0034] It has been found through research that the Kluyveromyces marxianus strain YZC-01 provided by the present invention for biodegradation of uric acid and the enzyme produced are safe for the human body, and both can biodegrade uric acid, having important application prospects in efficiently removing uric acid for the treatment of human hyperuricemia and corresponding complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] To make the present invention easy to understand, the present invention will be described below with reference to the accompanying drawings.

[0036] Figure 1 Shows the molecular phylogenetic tree of Kluyveromyces marxianus YZC-01 based on 18S rDNA.

[0037] Figure 2 Is the kinetic curve of uric acid biodegradation by Kluyveromyces marxianus YZC-01.

[0038] Figure 3 Is the kinetic curve of enzymatic degradation of uric acid by Kluyveromyces marxianus YZC-01.

[0039] Strain preservation

[0040] Kluyveromyces marxianus, isolated and identified by Beijing Beike Yiran Biotechnology Co., Ltd., has been deposited in the China General Microbiological Culture Collection Center (abbreviation: CGMCC; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit date: January 4, 2024, and the deposit number: CGMCC No. 29514. In the present invention, this strain is named Kluyveromyces marxianus strain YZC-01, also known as Kluyveromyces marxianus YZC-01. DETAILED DESCRIPTION OF THE INVENTION

[0041] To make the present invention easy to understand, the present invention 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 terms used herein are only for describing the specific embodiments and do not indicate any limitation.

[0042] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. 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.

[0043] Ⅰ. Terms

[0044] As used herein, the term "bacterial cells" refers to live and / or dead cells of Kluyveromyces marxianus.

[0045] As used herein, the term "crude enzyme" refers to a cell-free extract obtained by centrifuging the supernatant after disrupting the bacterial cells of Kluyveromyces marxianus.

[0046] As used herein, the term "pure crude enzyme" relative to the crude enzyme refers to a cell-free extract obtained by centrifuging the supernatant after disrupting the pure bacterial cells of Kluyveromyces marxianus.

[0047] As used herein, the term "microbial preparation" refers to preparations in various forms that are made from microorganisms with medical research value using traditional or modern biotechnology and act on the prevention (health care), treatment, and diagnosis of various physiological symptoms in the human body.

[0048] As used herein, the term "edible starch" refers to starch that meets the "National Standard for Edible Starch" (GB 31637-2016 National Food Safety Standard for Edible Starch).

[0049] The "water" used in the medium or fermentation culture process of the invention, unless otherwise specified, refers to sterile pure water obtained by filtering through a 0.22 μm filter membrane.

[0050] Ⅱ. Embodiments

[0051] As mentioned above, the existing dietary and drug therapies for controlling uric acid in the human body have slow and insignificant food and drug efficacy and have side effects. Therefore, the present inventors have conducted a large number of in-depth studies on the biodegradation of uric acid.

[0052] The present inventors have noticed that although lactic acid bacteria can promote intestinal health and reduce the accumulation of uric acid to some extent through a series of metabolic regulations, there are no reports on the direct and highly efficient biodegradation of uric acid by the strains used.

[0053] In recent years, the research on the human microbiome has been a hot topic and frontier in life science research. Although domestic and foreign scholars have screened microbial strains from traditional foods that can inhibit uric acid synthesis and degrade uric acid precursors such as inosine and guanosine, no evidence has been found that Kluyveromyces marxianus can directly and highly efficiently biodegrade uric acid.

[0054] The present inventor also noticed that Kluyveromyces marxianus belongs to eukaryotic microorganisms and is a yeast that can be directly consumed by humans approved by the National Health Commission of China. Although Kluyveromyces marxianus can promote human intestinal health and improve human immune function and has been widely used in the fields of food, fermentation, beer, and beverage production, no research reports on its biodegradation of uric acid have been found so far.

[0055] Based on long-term research on microorganisms, the present inventor successfully screened a strain of Kluyveromyces marxianus YZC-01 from the intestinal flora of healthy humans. Through research and exploration, the inventor found that the cells of this strain and the enzymes produced can degrade all of the uric acid with an initial concentration of 2 g / L within 72 h and 24 h, respectively. This not only has very important research value but also has important application prospects in the efficient biodegradation and removal of uric acid. Thus, the present invention was obtained.

[0056] Therefore, the Kluyveromyces marxianus strain YZC-01 for biodegradation of uric acid according to the first aspect of the present invention can produce enzymes that catalyze the degradation of uric acid.

[0057] The present inventor first successfully screened a strain of Kluyveromyces marxianus YZC-01 from the intestinal flora of healthy humans. By extracting genomic DNA, through PCR amplification and molecular identification based on 18S rDNA sequencing, it was determined to be Kluyveromyces marxianus. Based on the above, this strain was identified and named Kluyveromyces marxianus strain YZC-01. This strain has been deposited in the China General Microbiological Culture Collection Center, deposit number: CGMCC No. 29514.

[0058] The present inventor's research found that during the fermentation culture of Kluyveromyces marxianus strain YZC-01, the yeast cells produced during the fermentation culture contain more than one enzyme that can catalyze the degradation of uric acid. In the present invention, the mixture of these enzymes is called crude enzyme or Kluyveromyces marxianus YZC-01 crude enzyme.

[0059] Further research found that after centrifuging the cell-free lysate obtained by breaking the yeast cells, the supernatant cell-free extract was taken as Kluyveromyces marxianus YZC-01 crude enzyme. Thus, it is easy to understand that both the cells and the crude enzyme of Kluyveromyces marxianus strain YZC-01 can catalyze the degradation of uric acid.

[0060] The research results show that the Kluyveromyces marxianus strain YZC-01 can completely degrade and remove uric acid with an initial concentration of 2 g / L within 72 hours at an initial cell concentration of 2×10 7 / mL.

[0061] The crude enzyme produced by the Kluyveromyces marxianus strain YZC-01 can completely degrade uric acid with an initial concentration of 2 g / L within 24 hours at a protein concentration of 8.0 mg / mL.

[0062] Based on the above, the second to fourth aspects of the present invention further provide the uses or applications of the Kluyveromyces marxianus for biodegradation of uric acid described in the first aspect of the present invention.

[0063] Specifically, the second aspect of the present invention provides a Kluyveromyces marxianus strain YZC-01 preparation for biodegradation of uric acid, which belongs to a microbial preparation for biodegradation of uric acid and contains the bacterial cells and / or crude enzyme of the Kluyveromyces marxianus strain YZC-01 described in the first aspect of the present invention.

[0064] In some preferred embodiments of the present invention, the Kluyveromyces marxianus strain YZC-01 preparation contains the bacterial cells of the Kluyveromyces marxianus strain YZC-01 described in the first aspect of the present invention.

[0065] According to some embodiments of the present invention, the Kluyveromyces marxianus strain YZC-01 preparation for biodegradation of uric acid is a liquid preparation.

[0066] In some embodiments of the present invention, in the liquid preparation for biodegradation of uric acid, the cell concentration of the bacterial cells of the Kluyveromyces marxianus strain YZC-01 is (2-5)×10 7 / mL.

[0067] In some other embodiments of the present invention, in the liquid preparation for biodegradation of uric acid, the protein concentration of the crude enzyme of the Kluyveromyces marxianus strain YZC-01 is 5-10 mg / mL.

[0068] According to some other embodiments of the present invention, the Kluyveromyces marxianus strain YZC-01 preparation for biodegradation of uric acid is a solid powder preparation.

[0069] In some embodiments of the present invention, in the solid powder preparation for biodegradation of uric acid, the cell content of the bacterial cells of the Kluyveromyces marxianus strain YZC-01 is (1-6)×10 7 / g, preferably (2-5)×10 7 / g.

[0070] In other embodiments of the present invention, in the solid powder preparation for biodegradation of uric acid, the protein content of the crude enzyme of Kluyveromyces marxianus strain YZC-01 is 5-10 mg / g, preferably 8-10 mg / g.

[0071] The third aspect of the present invention provides a method for preparing a Kluyveromyces marxianus YZC-01 preparation for biodegradation of uric acid as described in the second aspect of the present invention, which includes:

[0072] Step B: Inoculate the fermentation strain into a fermentation medium, and perform fermentation culture at 18-40 °C, preferably 30-35 °C, with a shaking speed of 100-300 revolutions per minute for 3-5 days to obtain a fermentation culture of Kluyveromyces marxianus strain YZC-01;

[0073] Step C: Perform centrifugal separation on the fermentation culture of Kluyveromyces marxianus strain YZC-01 to harvest the bacterial cells of Kluyveromyces marxianus strain YZC-01;

[0074] Among them, the fermentation strain is obtained by seed culture from the corresponding Kluyveromyces marxianus strain YZC-01.

[0075] As is known to those skilled in the art, currently, 18S rRNA is commonly used internationally for molecular identification of eukaryotic microorganisms. Therefore, 18S rRNA can be used for comparison to obtain its homology in similarity comparison. Therefore, the fermentation strain used in the present invention is not limited to the wild isolate used in the present invention. 18S rDNA is the DNA sequence corresponding to the rRNA encoded on the bacterial chromosome and exists in the chromosomal genomes of all eukaryotic microorganisms. Figure 1 The molecular phylogenetic tree of Kluyveromyces marxianus strain YZC-01 based on 18S rDNA is shown.

[0076] In the above step C, the centrifugal separation treatment includes the precipitate obtained by centrifugally separating the liquid fermentation culture (i.e., the bacterial cells of Kluyveromyces marxianus strain YZC-01), which is resuspended and washed with physiological saline, and then centrifugally separated to obtain the bacterial cells of Kluyveromyces marxianus strain YZC-01.

[0077] The present invention does not particularly limit the conditions for centrifugal separation in the above step C. In some embodiments of the present invention, for example, the separation object can be centrifuged at 8000-10000 r / min for 10 min.

[0078] According to the method of the present invention, the fermentation culture is a shaking table or fermenter fermentation culture of the strain, and the fermentation strain is inoculated into the fermentation medium in the form of a seed solution. The inoculation amount of the seed solution is 0.1%-1% (v / v); preferably, the inoculation amount of the seed solution is 0.2%-0.5% (v / v); more preferably, the inoculation amount of the seed solution is 0.5% (v / v).

[0079] Specifically, based on 1 L of water, the above fermentation medium comprises the following components in 1 L of water:

[0080] Peptone 5-10 g;

[0081] Yeast powder 5-10 g; and

[0082] Glucose 3-8 g.

[0083] Preferably, based on 1 L of water, the above fermentation medium comprises the following components in 1 L of water:

[0084] Peptone 8-10 g;

[0085] Yeast powder 8-10 g; and

[0086] Glucose 4-6 g.

[0087] In some embodiments of the present invention, a 40% (wt / v) sodium hydroxide solution and a 36% (v / v) hydrochloric acid solution are used to adjust the initial pH of the fermentation medium to 6-8.

[0088] According to some embodiments of the present invention, the preparation method of the Kluyveromyces marxianus YZC-01 preparation of the present invention further comprises step A of seed culture before step B: picking a monoclonal colony of the Kluyveromyces marxianus YZC-01 strain provided by the present invention and inoculating it into 100 mL of a fermentation liquid medium, and culturing it on a shaking table at a temperature of 30°C and a rotation speed of 200 r / min for 3 days to obtain a fermentation strain (seed solution).

[0089] 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°C.

[0090] According to some embodiments of the present invention, the preparation method further comprises:

[0091] Step K, subjecting the cell suspension of the Kluyveromyces marxianus YZC-01 strain to cell disruption treatment in an ice-water bath (i.e., an ice-water mixture, 0-4°C) to obtain a cell-free lysate of the Kluyveromyces marxianus YZC-01 strain;

[0092] Step L: Centrifuge the cell-free lysate of Kluyveromyces marxianus YZC-01 strain, and take the supernatant cell-free extract as the crude enzyme of Kluyveromyces marxianus YZC-01 strain.

[0093] The present invention has no particular limitation on the conditions of the centrifugation separation in the above step L. In some embodiments of the present invention, for example, the material to be separated can be centrifuged at 15,000 - 18,000 r / min for 10 - 20 min.

[0094] The fourth aspect of the present invention provides an application of the Kluyveromyces marxianus YZC-01 preparation for biodegradable uric acid described in the second aspect of the present invention or the Kluyveromyces marxianus YZC-01 preparation for biodegradable uric acid prepared by the preparation method described in the third aspect of the present invention in the preparation of anti-hyperuricemic agents, which includes:

[0095] Step D: Wash the bacterial cells of Kluyveromyces marxianus YZC-01 strain with physiological saline to obtain pure bacterial cells of Kluyveromyces marxianus YZC-01 strain;

[0096] Step E: In a physiological saline system, under the low temperature condition of 0℃ - 4℃, ultrasonically disrupt the pure bacterial cells of Kluyveromyces marxianus YZC-01 strain, and take the supernatant after centrifugation to obtain a cell-free extract as the pure crude enzyme of Kluyveromyces marxianus YZC-01 strain;

[0097] Step F: Lyophilize the pure bacterial cells and / or pure crude enzyme of Kluyveromyces marxianus YZC-01 strain, and dilute the lyophilized Kluyveromyces marxianus YZC-01 preparation to prepare an anti-hyperuricemic agent.

[0098] In some embodiments of the present invention, in step F, the lyophilized Kluyveromyces marxianus YZC-01 preparation is diluted with physiological saline to prepare a liquid anti-hyperuricemic agent.

[0099] In other embodiments of the present invention, in step F, the lyophilized Kluyveromyces marxianus YZC-01 preparation is diluted with edible starch to prepare a solid anti-hyperuricemic agent.

[0100] In some preferred embodiments of the present invention, the anti-hyperuricemic agent is an oral preparation.

[0101] Ⅲ. Related materials and detection methods in the present invention

[0102] 1. Materials

[0103] The fecal microbiota capsule samples of healthy human intestines involved in the present invention were purchased from Beijing Fumat Biotechnology Co., Ltd., and permission for research and development activities was obtained.

[0104] 2. Detection method

[0105] (1) In the present invention, the cell concentration is measured by the following method:

[0106] For the determination of the cell concentration of Kluyveromyces marxianus YZC-01, take the culture of Kluyveromyces marxianus YZC-01, dilute it by a certain multiple with physiological saline, and directly measure the cell concentration therein using a flow cytometer (SYSMEX, Germany).

[0107] (2) In the present invention, the uric acid concentration is measured by the following method:

[0108] The uric acid concentration is measured using a high performance liquid chromatograph (Shimadzu LC-20AT). Specifically, the chromatographic column is Kromasil C18 (4.6×250 mm, 5 μm particle size); the mobile phase is methanol: 0.5% acetic acid aqueous solution (10:90); the detection wavelength is 283 nm; the flow rate is 1 mL / min; the injection volume is 20 μL; the temperature is 35°C.

[0109] (3) In the present invention, the crude enzyme protein concentration is measured by the following method:

[0110] Take the cell-free extract of Kluyveromyces marxianus YZC-01, dilute it by a certain multiple with phosphate buffer solution, add Coomassie Brilliant Blue G-250 dye reagent in proportion and react for 10 minutes, measure the absorbance at 595 nm using a 722S visible spectrophotometer (Shanghai Lingguang), and calculate the protein concentration by the standard curve method.

[0111] III. Examples

[0112] The present invention is specifically described below through specific examples. The experimental methods described below are all conventional laboratory methods unless otherwise specified. The experimental materials described below can be obtained from commercial channels unless otherwise specified.

[0113] Example 1:

[0114] (1) Prepare the growth medium for Kluyveromyces marxianus YZC-01, and its composition is (per liter): peptone 10.0 g, yeast extract 10.0 g, glucose 5.0 g. Add 100 mL of the prepared liquid medium to a 500 mL Erlenmeyer flask, sterilize it at high temperature and high pressure (121°C) for 20 minutes, and then disinfect it under ultraviolet irradiation in a clean workbench for another 20 minutes.

[0115] (2) Under aseptic conditions in a clean bench, inoculate 0.5 mL of Kluyveromyces marxianus YZC-01 bacterial liquid into a triangular flask liquid medium. After batch culturing for 3 days at a temperature of 30 °C and a shaker speed of 200 rpm, harvest Kluyveromyces marxianus YZC-01 cells by centrifuging (8000 rpm, 10 minutes) and then pouring off the supernatant.

[0116] Take 20 mL of the Kluyveromyces marxianus YZC-01 cell suspension and add it to a 50 mL glass tube, then insert it into ice water. Use an ultrasonic cell disruptor to break the Kluyveromyces marxianus YZC-01 cells. The conditions are: ultrasonic power 400 W, interval 2 seconds, ultrasonic oscillation 10 seconds, and disruption time 15 minutes (5 minutes each time). After cell disruption, 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 Kluyveromyces marxianus YZC-01.

[0117] (3) According to different concentrations of uric acid, add the prepared Kluyveromyces marxianus YZC-01 cells and crude enzyme as a rapid, safe, and efficient biocatalyst in a certain proportion to achieve the purpose of rapidly and efficiently degrading and removing uric acid.

[0118] Figure 1 It shows that the strain we screened has the closest genetic relationship with Kluyveromyces marxianus, so it is named Kluyveromyces marxianus YZC-01 strain.

[0119] Figure 2 It shows that at an initial cell concentration of 2.0×10 7 / mL of Kluyveromyces marxianus YZC-01, uric acid with an initial concentration of 2 g / L can be completely degraded within 72 hours, indicating that Kluyveromyces marxianus YZC-01 has a strong biodegradation ability for uric acid.

[0120] Figure 3 It shows that the cell-free extract (crude enzyme) of Kluyveromyces marxianus YZC-01 can catalyze the degradation of uric acid at a faster rate. At a protein concentration of 8.0 mg / mL, uric acid with an initial concentration of 2 g / L can be completely degraded within 24 hours, having a higher uric acid degradation rate.

[0121] It should be noted that the above-described embodiments are only preferred embodiments of the present invention for explaining the present invention and do not constitute any limitation to the present invention. The present invention has been described by reference to exemplary embodiments, but it should be understood that the words used therein are descriptive and explanatory terms rather than limiting terms. Modifications may be made to the present invention within the scope of the claims of the present invention as provided, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A Kluyveromyces marxianus yeast that degrades uric acid Kluyveromyces marxianus ) Use of the YZC-01 strain in the preparation of a uric acid-lowering agent, comprising: Step D, washing the bacterial cells of Kluyveromyces marxianus YZC-01 strain with physiological saline to obtain pure bacterial cells of Kluyveromyces marxianus YZC-01 strain; Step E, in a physiological saline system, under low temperature conditions, using ultrasound to disrupt the pure bacterial cells of Kluyveromyces marxianus YZC-01 strain, and taking the supernatant after centrifugation to obtain a cell-free extract as a crude enzyme pure product of Kluyveromyces marxianus YZC-01 strain; Step F, freeze-drying the pure bacterial cell product and / or the pure crude enzyme product of Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare a uric acid-degrading agent; Wherein, the temperature of the low temperature is 0-4°C; The uric acid-degrading Kluyveromyces marxianus YZC-01 strain can produce an enzyme that catalyzes the degradation of uric acid. The Kluyveromyces marxianus YZC-01 strain has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.29514.

2. The use according to claim 1, characterized in that: The Kluyveromyces marxianus YZC-01 strain was prepared at an initial bacterial cell concentration of 2×10 7 / mL, the initial concentration of uric acid of 2 g / L can be completely degraded and removed within 72 hours; The crude enzyme produced by the Kluyveromyces marxianus YZC-01 strain can completely remove uric acid with an initial concentration of 2 g / L within 24 hours at a protein concentration of 8.0 mg / mL.

3. The use according to claim 1, characterized in that: In step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with physiological saline to prepare a liquid uric acid-lowering agent; In the liquid uric acid-lowering agent, the bacterial cell concentration of Kluyveromyces marxianus YZC-01 strain is (2-5)×10 7 / mL; and / or, in the liquid uric acid-lowering agent, the protein concentration of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 5-10 mg / mL.

4. The use according to claim 1, characterized in that: In step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with edible starch to prepare a solid uric acid-lowering agent; In the solid uric acid-lowering agent, the content of bacterial cells of Kluyveromyces marxianus YZC-01 strain is (2-5)×10 7 / g; and / or, in the solid uric acid-lowering agent, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 8-10 mg / g.

5. The use according to any one of claims 1 to 4, characterized in that: The method for preparing bacterial cells of the Kluyveromyces marxianus YZC-01 strain comprises: Step B, inoculating the fermentation bacteria into a fermentation medium for fermentation culture to obtain a fermentation culture of Kluyveromyces marxianus YZC-01 strain; Step C, centrifuging the fermentation culture of Kluyveromyces marxianus YZC-01 strain to harvest bacterial cells of Kluyveromyces marxianus YZC-01 strain; The fermentation strain is obtained by seed culture of the corresponding Kluyveromyces marxianus YZC-01 strain.

6. The use according to claim 5, characterized in that: The fermentation medium, based on 1L of water, includes the following components in 1L of water: Peptone 5-10g; Yeast powder 5-10g; as well as Glucose 3-8g; The pH value of the fermentation medium is 7-8; In step B, the fermentation culture temperature is 30-35°C.

7. The use according to any one of claims 1 to 4, characterized in that: The uric acid lowering agent is an oral preparation.

8. The use according to claim 5, characterized in that: The uric acid lowering agent is an oral preparation.

9. The use according to claim 6, characterized in that: The uric acid lowering agent is an oral preparation.

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