L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation and its application

Abstract

The present invention belongs to the field of biotechnology and relates to a Kluyveromyces marxianus YZC-01 strain for biodegrading L-homocysteine, which is capable of producing an enzyme that catalyzes the degradation of L-homocysteine. The present invention also relates to a Kluyveromyces marxianus YZC-01 preparation for degrading L-homocysteine, which contains bacterial cells and / or crude enzymes of the above-mentioned Kluyveromyces marxianus YZC-01 strain. Research results show that the Kluyveromyces marxianus YZC-01 strain for biodegrading L-homocysteine ​​provided by the present invention and the enzyme produced therefrom are safe for the human body and are both capable of efficiently biodegrading L-homocysteine, and have important application prospects in removing L-homocysteine ​​for treating human hyper-L-homocysteinemia and corresponding complications.

Description

Technical Field

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

[0002] In recent years, the incidence of cardiovascular and cerebrovascular diseases has increased year by year. The main reason is that atherosclerosis, thrombosis and the formation of plaques hinder the flow of blood, leading to myocardial infarction or cerebral infarction. It is the disease with the highest mortality rate in my country and even in the world.

[0003] L-homocysteine ​​(L-4,4'-dithiobis(2-aminobutyric acid, molecular formula: C8H 16 L-homocysteine ​​(N2O4S2, molecular weight: 268.35), also known as homocysteine, is a sulfur-containing amino acid and a major risk factor for cardiovascular and cerebrovascular diseases. Plasma L-homocysteine ​​levels exceeding 15 μmol / L are harmful. L-homocysteine ​​can not only cause atherosclerosis, hypertension, and coronary heart disease, but can also induce aortic venous thrombosis, leading to myocardial infarction and cerebral infarction. Its mechanism of action is that L-homocysteine ​​damages vascular endothelial cells, causing proliferation of vascular smooth muscle cells and obstructing blood flow. It can also enhance platelet function and promote thrombus formation.

[0004] Currently, treatments for hyperhomocysteinemia include dietary therapy and medication. Dietary therapy primarily involves supplementing with nutrients like B vitamins and folic acid, while reducing the intake of methionine-rich foods like meat and cheese. Medication, including medications like nebivolol and rosiglitazone, is often ineffective and associated with adverse reactions such as headaches, fatigue, hypotension, and bradycardia. Consequently, existing medications or foods for controlling L-homocysteinemia are ineffective and have side effects. Therefore, further research is needed into effective treatments or methods for hyperhomocysteinemia. Summary of the Invention

[0005] One of the purposes of the present invention is to address the problems in the prior art of drugs or foods for degrading and removing L-homocysteine, which are ineffective and have side effects. The present invention provides a Kluyveromyces marxianus YZC-01 strain for biodegrading L-homocysteine. The bacterial cells of the strain and the enzymes produced by the strain are safe for the human body and can efficiently biodegrade L-homocysteine, thus having important application prospects in removing L-homocysteine.

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

[0007] To this end, the first aspect of the present invention provides a Kluyveromyces marxianus YZC-01 strain for biodegrading L-homocysteine, which can produce an enzyme that catalyzes the degradation of L-homocysteine ​​and has a deposit number of CGMCC No. 29514.

[0008] In some embodiments of the present invention, the bacterial cells of the Kluyveromyces marxianus strain YZC-01 can completely degrade and remove L-homocysteine ​​at an initial concentration of 500 mg / L within 48 hours.

[0009] In other embodiments of the present invention, the crude enzyme of Kluyveromyces marxianus strain YZC-01 can completely remove L-homocysteine ​​with an initial concentration of 500 mg / L within 24 hours at a protein concentration of 3.2 mg / mL.

[0010] The second aspect of the present invention provides a Kluyveromyces marxianus YZC-01 preparation for degrading L-homocysteine, which contains bacterial cells and / or crude enzymes 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 L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation is a liquid preparation; preferably, in the L-homocysteine-degrading liquid preparation, the concentration of Kluyveromyces marxianus YZC-01 bacterial cells is (2-5)×10 7 / mL; and / or, in the liquid preparation for degrading L-homocysteine, the protein concentration of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 3-10 mg / mL.

[0012] In other embodiments of the present invention, the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation is a solid powder preparation; preferably, in the L-homocysteine-degrading solid powder preparation, the content of bacterial cells of the Kluyveromyces marxianus YZC-01 strain is (1-6)×10 7 / g, more preferably (2-5)×10 7 / g; and / or, in the solid powder preparation for degrading L-homocysteine, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 10-60 mg / g, more preferably 20-40 mg / g.

[0013] The third aspect of the present invention provides a method for preparing the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation according to the second aspect of the present invention, comprising:

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

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

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

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

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

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

[0020] Preferably, the pH value of the fermentation medium is 6-7;

[0021] Further preferably, in step B, the fermentation culture temperature is 18-40°C, preferably 28-32°C.

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

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

[0024] Step L, centrifuging the cell-free disrupted liquid 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 low temperature is 0°C-4°C.

[0026] The fourth aspect of the present invention provides a use of the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation according to the second aspect of the present invention or the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation prepared by the preparation method according to the third aspect of the present invention in the preparation of an L-homocysteine-lowering agent, comprising:

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

[0028] Step E: in a physiological saline system, at low temperature, using ultrasonication to disrupt the pure bacterial cells of Kluyveromyces marxianus YZC-01 strain, centrifuging the cells, and collecting the supernatant to obtain a cell-free extract as a crude enzyme of Kluyveromyces marxianus YZC-01 strain;

[0029] Step F, freeze-drying the bacterial cells and / or crude enzyme of the Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare an L-homocysteine-lowering agent;

[0030] Wherein, the low temperature is 0°C-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 L-homocysteine-lowering agent.

[0032] In other embodiments of the present invention, in step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with edible starch to prepare a solid L-homocysteine-lowering agent.

[0033] In some preferred embodiments of the present invention, the L-homocysteine-lowering agent is an oral preparation.

[0034] Studies have found that the Kluyveromyces marxianus YZC-01 strain for biodegrading L-homocysteine ​​and the enzyme produced by the present invention are safe for the human body and can biodegrade L-homocysteine. They have important application prospects in the efficient removal of L-homocysteine ​​for the treatment of human hyper-L-homocysteinemia and its corresponding complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to make the present invention easier to understand, the present invention is described below with reference to the accompanying drawings.

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

[0037] Figure 2 This is the kinetic curve of biodegradation of L-homocysteine ​​by Kluyveromyces marxianus YZC-01.

[0038] Figure 3 This is the kinetic curve of L-homocysteine ​​degradation catalyzed by Kluyveromyces marxianus YZC-01 enzyme.

[0039] strain collection

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

[0041] To facilitate understanding of the present invention, 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 terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[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 the present 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, preferred methods and materials are now described.

[0043] I. Terminology

[0044] The term "bacteria" as used in the present invention refers to living and / or dead cells of Kluyveromyces marxianus.

[0045] The term "crude enzyme" as used in the present invention refers to a cell-free extract obtained by disrupting Kluyveromyces marxianus cells and then centrifuging the supernatant.

[0046] The term "pure crude enzyme" in the present invention refers to a cell-free extract obtained by breaking the pure bacterial cells of Kluyveromyces marxianus and then centrifuging the supernatant.

[0047] The term "microbial preparation" as used in the present invention refers to various forms of preparations that are made from microorganisms with medical research value using traditional or modern biotechnology to prevent (health care), treat and diagnose various physiological symptoms of the human body.

[0048] The term "edible starch" as used in the present invention refers to starch that meets the "National Standard for Edible Starch" (GB 31637-2016 National Food Safety Standard Edible Starch).

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

[0050] II. Implementation Plan

[0051] As mentioned above, existing diet and drug therapies for controlling L-homocysteine ​​in the human body are slow and ineffective and have side effects. Therefore, the present inventors have conducted extensive research on the biodegradation of L-homocysteine.

[0052] The present inventors noted that although lactic acid bacteria can promote intestinal health and reduce the accumulation of L-homocysteine ​​to some extent through a series of metabolic regulation, there is no report on the bacteria used to directly and efficiently biodegrade L-homocysteine.

[0053] The inventors also noted that Kluyveromyces marxianus, a eukaryotic microorganism, is the only yeast approved for direct human consumption by my country's National Health Commission. While Kluyveromyces marxianus can promote intestinal health and enhance human immunity, and is widely used in food, fermentation, beer, and beverage production, no research has yet been found on its ability to biodegrade L-homocysteine.

[0054] Based on long-term microbial research, the inventors successfully isolated a pure strain of microorganisms from healthy human intestinal bacteria that can efficiently biodegrade L-homocysteine. This strain and the enzyme it produces are both capable of efficiently biodegrading L-homocysteine, demonstrating not only significant research value but also promising application prospects in the efficient biodegradation and removal of L-homocysteine. This led to the present invention.

[0055] Therefore, the Kluyveromyces marxianus YZC-01 strain for biodegrading L-homocysteine ​​according to the first aspect of the present invention can produce an enzyme that catalyzes the degradation of L-homocysteine.

[0056] The inventors first successfully isolated a strain of Kluyveromyces marxianus, YZC-01, from the intestinal flora of healthy humans. Genomic DNA was extracted, amplified by PCR, and molecularly identified using 18S rDNA sequencing. Based on this, the strain was identified and named Kluyveromyces marxianus strain YZC-01. This strain has been deposited with the General Microbiology Center of the China General Culture Collection Administration under the accession number CGMCC No. 29514.

[0057] The present inventors have discovered that yeast cells produced during fermentation of Kluyveromyces marxianus YZC-01 contain more than one enzyme capable of catalyzing the degradation of L-homocysteine. In the present invention, the mixture of these enzymes is referred to as crude enzyme, or Kluyveromyces marxianus YZC-01 crude enzyme.

[0058] Further research revealed that the cell-free lysate after yeast cell disruption was centrifuged and the supernatant, a cell-free extract obtained as crude Kluyveromyces marxianus YZC-01 enzyme, was able to catalyze the degradation of L-homocysteine.

[0059] The research results show that the bacterial cells of the Kluyveromyces marxianus YZC-01 strain can completely degrade and remove L-homocysteine ​​with an initial concentration of 500 mg / L within 48 hours.

[0060] The crude enzyme produced by the Kluyveromyces marxianus YZC-01 strain can completely degrade L-homocysteine ​​with an initial concentration of 500 mg / L within 24 hours at a protein concentration of 3.2 mg / mL.

[0061] Based on the above, the second to fourth aspects of the present invention further provide uses or applications of the Kluyveromyces marxianus for biodegrading L-homocysteine ​​according to the first aspect of the present invention.

[0062] Specifically, the second aspect of the present invention provides a Kluyveromyces marxianus YZC-01 preparation for biodegrading L-homocysteine, which is a microbial preparation for biodegrading L-homocysteine, and contains bacterial cells and / or crude enzymes of the Kluyveromyces marxianus YZC-01 strain as described in the first aspect of the present invention.

[0063] In some preferred embodiments of the present invention, the Kluyveromyces marxianus YZC-01 preparation contains bacterial cells of the Kluyveromyces marxianus YZC-01 strain as described in the first aspect of the present invention.

[0064] According to some embodiments of the present invention, the L-homocysteine ​​biodegrading Kluyveromyces marxianus YZC-01 preparation is a liquid preparation.

[0065] In some embodiments of the present invention, the bacterial cell concentration of Kluyveromyces marxianus YZC-01 strain in the liquid preparation for biodegradation of L-homocysteine ​​is (2-5)×10 7 / mL.

[0066] In other embodiments of the present invention, in the liquid preparation for biodegrading L-homocysteine, the protein concentration of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 3-10 mg / mL.

[0067] According to other embodiments of the present invention, the L-homocysteine ​​biodegrading Kluyveromyces marxianus YZC-01 preparation is a solid powder preparation.

[0068] In some embodiments of the present invention, in the solid powder preparation for biodegradation of L-homocysteine, the bacterial cell content of Kluyveromyces marxianus YZC-01 strain is (1-6)×10 7 / g, preferably (2-5)×10 7 / g.

[0069] In other embodiments of the present invention, in the solid powder preparation for biodegrading L-homocysteine, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 10-60 mg / g, preferably 20-40 mg / g.

[0070] The third aspect of the present invention provides a method for preparing the L-homocysteine ​​biodegrading Kluyveromyces marxianus YZC-01 preparation according to the second aspect of the present invention, comprising:

[0071] Step B, inoculating the fermentation bacteria into a fermentation medium, and fermenting and culturing at 18-40° C., preferably 25-30° C., and a shaking speed of 100-300 rpm for 3-5 days to obtain a fermentation culture of Kluyveromyces marxianus YZC-01 strain;

[0072] Step C, centrifuging the fermentation culture of Kluyveromyces marxianus YZC-01 to harvest the bacterial cells of Kluyveromyces marxianus YZC-01;

[0073] The fermentation strain is obtained by seed culture of the corresponding Kluyveromyces marxianus YZC-01 strain.

[0074] As those skilled in the art are aware, 18S rRNA is currently commonly used internationally for molecular identification of eukaryotic microorganisms. Therefore, 18S rRNA can be used for comparison and homology analysis. Therefore, the fermentation strains used in the present invention are not limited to the field isolates used in the present invention. 18S rDNA is the DNA sequence on bacterial chromosomes that encodes rRNA and is present in the chromosomal genomes of all eukaryotic microorganisms. Figure 1 The molecular evolutionary tree of Kluyveromyces marxianus strain YZC-01 based on 18S rDNA is shown.

[0075] In the above step C, the centrifugation treatment includes resuspending and washing the precipitate obtained by centrifuging the liquid fermentation culture (i.e., the bacterial cells of Kluyveromyces marxianus YZC-01 strain) with physiological saline, and then centrifuging it to obtain the bacterial cells of Kluyveromyces marxianus YZC-01 strain.

[0076] The present invention has no particular limitation on the conditions for centrifugal separation in step C. In some embodiments of the present invention, for example, the material to be separated can be centrifuged at 8000-10000 r / min for 10 min.

[0077] According to the method of the present invention, the fermentation culture is a shaker or fermentation tank fermentation culture of the bacterial strain, and the fermentation bacterial 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, the inoculation amount of the seed liquid is 0.2%-0.5% (v / v); more preferably, the inoculation amount of the seed liquid is 0.5% (v / v).

[0078] Specifically, the fermentation medium comprises the following components in 1 L of water:

[0079] Peptone 5-10g;

[0080] 5-10g yeast powder; and

[0081] 3-8g of glucose.

[0082] Preferably, the fermentation medium comprises the following components in 1 L of water:

[0083] Peptone 8-10g;

[0084] 8-10g yeast powder; and

[0085] Glucose 4-6g.

[0086] In some embodiments of the present invention, the initial pH of the fermentation medium is adjusted to 6-7 using 40% (wt / v) sodium hydroxide solution and 36% (v / v) hydrochloric acid solution.

[0087] According to some embodiments of the present invention, the preparation method of the Kluyveromyces marxianus YZC-01 preparation involved in the present invention further includes a seed culture step A 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 fermentation liquid culture medium, and culturing it on a shaking table at a temperature of 28°C and a speed of 200 r / min for 3 days to obtain a fermentation strain (seed liquid).

[0088] The inventors studied the effects of different temperatures on the growth of Kluyveromyces marxianus YZC-01 and found that Kluyveromyces marxianus YZC-01 grew faster at a temperature of 28°C.

[0089] According to some embodiments of the present invention, the preparation method further comprises:

[0090] Step K, disrupting the cell suspension of the Kluyveromyces marxianus YZC-01 strain in an ice-water bath (i.e., an ice-water mixture, 0-4° C.) to obtain a cell-free disrupted solution of the Kluyveromyces marxianus YZC-01 strain;

[0091] In step L, the cell-free disrupted liquid of Kluyveromyces marxianus YZC-01 strain is centrifuged, and the supernatant cell-free extract is used as the crude enzyme of Kluyveromyces marxianus YZC-01 strain.

[0092] The present invention has no particular limitation on the conditions for centrifugal separation in 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.

[0093] The fourth aspect of the present invention provides a use of the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation according to the second aspect of the present invention or the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation prepared by the preparation method according to the third aspect of the present invention in the preparation of an L-homocysteine-lowering agent, comprising:

[0094] Step D, washing the Kluyveromyces marxianus YZC-01 cells with physiological saline to obtain pure Kluyveromyces marxianus YZC-01 cells;

[0095] Step E: in a physiological saline system, at a low temperature of 0°C to 4°C, using ultrasonication to disrupt the pure bacterial cells of Kluyveromyces marxianus YZC-01 strain, and after centrifugation, the supernatant is collected to obtain a cell-free extract as a crude enzyme pure product of Kluyveromyces marxianus YZC-01 strain;

[0096] Step F: freeze-drying the pure bacterial cells and / or crude enzyme of Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare an L-homocysteine-lowering agent.

[0097] 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 L-homocysteine-lowering agent.

[0098] In other embodiments of the present invention, in step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with edible starch to prepare a solid L-homocysteine-lowering agent.

[0099] In some preferred embodiments of the present invention, the L-homocysteine-lowering agent is an oral preparation.

[0100] III. Related Materials and Detection Methods in the Present Invention

[0101] 1. Materials

[0102] The healthy human intestinal fecal bacteria capsule samples involved in the present invention were purchased from Beijing Fumate Biotechnology Co., Ltd. and obtained permission to use them for scientific research activities.

[0103] 2. Detection method

[0104] (1) The cell concentration in the present invention is determined by the following method:

[0105] To determine the cell concentration of Kluyveromyces marxianus YZC-01, the culture of Kluyveromyces marxianus YZC-01 was diluted a certain multiple with physiological saline, and the cell concentration was directly determined using a flow cytometer (SYSMEX, Germany).

[0106] (2) The concentration of L-homocysteine ​​in the present invention is measured by the following method:

[0107] The method for determining the concentration of L-homocysteine ​​is to use a pure aqueous solution to fully dissolve the L-homocysteine ​​in the liquid culture medium in proportion, derivatize it with 9-fluorenylmethyl chloroformate derivatization reagent for 10 minutes, and then take an appropriate amount of supernatant after centrifugation. The L-homocysteine ​​concentration in the liquid culture medium is determined using a RID-20A high-performance liquid chromatograph (Shimadzu).

[0108] (3) The crude enzyme protein concentration in the present invention is measured by the following method:

[0109] The cell-free extract of Kluyveromyces marxianus YZC-01 was diluted a certain number of times with phosphate buffer solution, and 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.

[0110] III. Examples

[0111] The present invention is described in detail below through specific examples. The experimental methods described below, unless otherwise specified, are all routine laboratory methods. The experimental materials described below, unless otherwise specified, can all be obtained from commercial channels.

[0112] Example 1:

[0113] (1) Prepare a growth medium for Kluyveromyces marxianus YZC-01, consisting of (per liter): 10.0 g peptone, 10.0 g yeast powder, and 5.0 g glucose. Add 100 ml of the prepared liquid medium to a 500 ml Erlenmeyer flask, sterilize under high temperature and pressure (121°C) for 20 minutes, and then sterilize under ultraviolet light in a clean bench for another 20 minutes.

[0114] (2) Under sterile conditions in a clean bench, 0.5 ml of Kluyveromyces marxianus YZC-01 bacterial liquid was inoculated into a triangular flask liquid culture medium. After batch culture at 28°C and a shaker speed of 200 rpm for 3 days, the Kluyveromyces marxianus YZC-01 cells were harvested by centrifugation (8000 rpm, 10 minutes) and the supernatant was discarded.

[0115] 20 mL of Kluyveromyces marxianus YZC-01 cell suspension was added to a 50 mL glass tube and then plunged into ice water. The Kluyveromyces marxianus YZC-01 cells were disrupted using an ultrasonic cell disruptor under the following conditions: ultrasonic power of 400 W, 2-second intervals, 10-second ultrasonic oscillations, and a disruption time of 15 minutes (5 minutes per interval). After cell disruption, the cell suspension was centrifuged at 15,000 rpm for 20 minutes, and the supernatant was slowly decanted to obtain the Kluyveromyces marxianus YZC-01 cell-free extract (crude enzyme).

[0116] (3) According to the different concentrations of L-homocysteine, the cultured Kluyveromyces marxianus YZC-01 cells and crude enzymes were added in a certain proportion as a fast, safe and efficient biocatalyst to achieve the purpose of rapid and efficient degradation and removal of L-homocysteine.

[0117] Figure 1 The strain we screened showed that it was most closely related to Kluyveromyces marxianus, so it was named Kluyveromyces marxianus YZC-01.

[0118] Figure 2 It showed that within 48 hours, the concentration of Kluyveromyces marxianus YZC-01 cells increased from 1.0×10 6 / mL grew to 3.0×10 7 / mL, and could completely degrade L-homocysteine ​​with an initial concentration of 500 mg / L, indicating that Kluyveromyces marxianus YZC-01 had a strong biodegradation ability for L-homocysteine.

[0119] Figure 3 The results showed that the cell-free extract (crude enzyme) of Kluyveromyces marxianus YZC-01 could catalyze the degradation of L-homocysteine ​​at a faster rate. At a protein concentration of 3.2 mg / mL, L-homocysteine ​​with an initial concentration of 500 mg / L could be completely degraded within 24 hours, indicating a higher L-homocysteine ​​degradation rate.

[0120] It should be noted that the embodiments described above are only preferred embodiments of the present invention and are used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, 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 YZC-01 strain for biodegrading L-homocysteine, characterized in that: The Kluyveromyces marxianus YZC-01 strain can produce an enzyme that catalyzes the degradation of L-homocysteine, and its deposit number is CGMCC No. 29514.

2. A Kluyveromyces marxianus YZC-01 preparation for biodegrading L-homocysteine, characterized in that: The Kluyveromyces marxianus YZC-01 preparation contains bacterial cells of the Kluyveromyces marxianus YZC-01 strain according to claim 1 and / or crude enzymes of the Kluyveromyces marxianus YZC-01 strain.

3. The Kluyveromyces marxianus YZC-01 preparation according to claim 2, characterized in that The L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation is a liquid preparation.

4. The Kluyveromyces marxianus YZC-01 preparation according to claim 3, characterized in that In the liquid preparation for degrading L-homocysteine, the bacterial cell concentration of Kluyveromyces marxianus YZC-01 strain is (2-5)×10 7 / mL; and / or, in the liquid preparation for degrading L-homocysteine, the protein concentration of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 3-10 mg / mL.

5. The Kluyveromyces marxianus YZC-01 preparation according to claim 2, characterized in that The L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation is a solid powder preparation.

6. The Kluyveromyces marxianus YZC-01 preparation according to claim 5, characterized in that In the solid powder preparation for degrading L-homocysteine, the content of Kluyveromyces marxianus YZC-01 bacterial cells is (1-6)×10 7 / g; and / or, in the solid powder preparation for degrading L-homocysteine, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 10-60 mg / g.

7. The Kluyveromyces marxianus YZC-01 preparation according to claim 6, characterized in that In the solid powder preparation for degrading L-homocysteine, the content of Kluyveromyces marxianus YZC-01 bacterial cells is (2-5)×10 7 / g; and / or, in the solid powder preparation for degrading L-homocysteine, the protein content of the crude enzyme of Kluyveromyces marxianus YZC-01 strain is 20-40 mg / g.

8. A method for preparing the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation according to any one of claims 2 to 7, comprising: 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 to harvest the bacterial cells of Kluyveromyces marxianus YZC-01; The fermentation strain is obtained by seed culture of the corresponding Kluyveromyces marxianus YZC-01 strain.

9. The preparation method according to claim 8, characterized in that The fermentation medium, based on 1 L of water, includes the following components in 1 L of water: Peptone 5-10g; 5-10g yeast powder; as well as Glucose 3-8g; The pH value of the fermentation medium is 6-7.

10. The preparation method according to claim 9, characterized in that The fermentation medium, based on 1 L of water, includes the following components in 1 L of water: Peptone 8-10g; 8-10g yeast powder; as well as Glucose 4-6g.

11. The preparation method according to claim 9, characterized in that In step B, the fermentation culture temperature is 18-40°C.

12. The preparation method according to claim 11, characterized in that In step B, the fermentation temperature is 25-30°C.

13. The preparation method according to any one of claims 8 to 12, characterized in that: The preparation method further comprises: Step K, performing cell disruption treatment on the cell suspension of the Kluyveromyces marxianus YZC-01 strain under low temperature conditions to obtain a cell-free disrupted solution of the Kluyveromyces marxianus YZC-01 strain; Step L, centrifuging the cell-free disrupted liquid of Kluyveromyces marxianus YZC-01 strain, and taking the supernatant cell-free extract as the crude enzyme of Kluyveromyces marxianus YZC-01 strain; Wherein, the low temperature is 0°C-4°C.

14. Use of the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation according to any one of claims 2 to 7 or the L-homocysteine-degrading Kluyveromyces marxianus YZC-01 preparation obtained by the preparation method according to any one of claims 8 to 13 in the preparation of an L-homocysteine-lowering agent, comprising: Step D, washing the Kluyveromyces marxianus YZC-01 cells with physiological saline to obtain pure Kluyveromyces marxianus YZC-01 cells; Step E: in a physiological saline system, at low temperature, using ultrasonication to disrupt the pure bacterial cells of Kluyveromyces marxianus YZC-01 strain, centrifuging the cells, and collecting the supernatant to obtain a cell-free extract as a crude enzyme of Kluyveromyces marxianus YZC-01 strain; Step F, freeze-drying the pure bacterial cells and / or crude enzyme of Kluyveromyces marxianus YZC-01 strain, and diluting the freeze-dried Kluyveromyces marxianus YZC-01 preparation to prepare an agent for degrading L-homocysteine; Wherein, the low temperature is 0°C-4°C.

15. The use according to claim 14, characterized in that In step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with physiological saline to prepare a liquid L-homocysteine-lowering agent; Alternatively, in step F, the freeze-dried Kluyveromyces marxianus YZC-01 preparation is diluted with edible starch to prepare a solid L-homocysteine-lowering agent.

16. The use according to claim 15, characterized in that The L-homocysteine-lowering agent is an oral preparation.

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