WO2021142710A1 - 一种灵芝菌丝体降压肽及其制备方法 - Google Patents

一种灵芝菌丝体降压肽及其制备方法 Download PDF

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WO2021142710A1
WO2021142710A1 PCT/CN2020/072454 CN2020072454W WO2021142710A1 WO 2021142710 A1 WO2021142710 A1 WO 2021142710A1 CN 2020072454 W CN2020072454 W CN 2020072454W WO 2021142710 A1 WO2021142710 A1 WO 2021142710A1
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peptide
ganoderma lucidum
antihypertensive
fermentation
strain
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French (fr)
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刘高强
伍强
王永红
王晓玲
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中南林业科技大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to a ganoderma mycelium antihypertensive peptide and a preparation method thereof, and belongs to the technical fields of polypeptide biotechnology and antihypertensive drugs.
  • Hypertension is one of the main diseases threatening human health, and it is a common cardiovascular and cerebrovascular disease in life. With the development of modern society and the improvement of people's living standards, the incidence of hypertensive cardiovascular and cerebrovascular diseases has greatly increased, and it is becoming younger and younger. According to the statistics of the "2018 World Health Statistics Report", the death rate of cerebrovascular diseases in the non-communicable disease center is the first, with 17.9 million deaths, and the total number of patients with hypertension in my country has exceeded 300 million.
  • the recognized synthetic drugs for lowering blood pressure such as captopril, ramipril, and lisinopril have side effects such as fatigue, nausea, severe cough, and rash and itching during clinical application.
  • Angiotensin I converting enzyme is a vascular endothelial cell membrane-bound enzyme. It catalyzes the conversion of angiotensin I to angiotensin II to increase blood pressure and is an ideal target for the treatment of hypertension.
  • ACE inhibitory peptides are a class of small molecular substances with potential blood pressure lowering effects, which can inhibit ACE enzyme activity, reduce the damage of bradykinin and reduce the degradation of angiotensin I.
  • ACE inhibitory peptides have been obtained from animal products, marine organisms, and plants (Seung, et al. Antihypertensive peptides from animal products, marine organisms, and plans-a review.
  • the components of the enzymatic hydrolysis product obtained by the directed enzymatic hydrolysis technology are relatively complex, and subsequent purification is difficult; the enzymatic hydrolysis process needs to strictly control the enzymatic conditions of the protease (addition of enzyme, temperature, pH, etc.), which will consume more "material + energy” +Manpower", the production cost is high, which restricts the development of large-scale production of ACE inhibitory peptides.
  • a few foreign researchers have discovered natural ACE inhibitory peptides from traditional fermentation products, opening up a new idea of using microorganisms to prepare natural ACE inhibitory peptides.
  • ACE inhibitory peptides including cuttlefish muscle protein Val-Glu-Leu-Tyr-Pro, egg His-Leu-Phe-Gly-Pro-Pro-Gly-Lys-Lys-Asp-Pro-Val, silkworm pupa Gly-Ala -Met-Val-Val-His, peanut Tyr-Leu-Val-Arg and other peptides from different sources, with different molecular weights, usually composed of 4-12 amino acid residues, although they have strong ACE inhibitory activity in vitro
  • most researchers have not verified its blood pressure lowering effect in vivo, and the mechanism of lowering blood pressure in vivo is still unclear.
  • Ganoderma is a fungus with high pharmacological value. It has many functions such as anti-tumor, regulating immunity, protecting liver and lowering blood pressure. Triterpenoids and polysaccharides are the two main active components of Ganoderma lucidum, but there are few reports on Ganoderma lucidum active oligopeptides.
  • Ganoderma lucidum fruit body has a long cultivation period (4-9 months) and is easily affected by the season and environment. Therefore, the biological stability of the active substance of the fruit body is poor. By separating the Ganoderma lucidum cells and using liquid fermentation technology, the Ganoderma lucidum mycelium is prepared To obtain active substances is a more effective means.
  • the development and utilization of Ganoderma lucidum mycelium as a new resource for the preparation of active peptides has important practical significance.
  • the inventor team obtained three ACE inhibitory peptides from Ganoderma lucidum mycelium, the amino acid sequences of which are Gln-Leu-Asp-Leu, Gln-Leu-Val-Pro and Gln-Asp-Val- Leu, the IC50 for inhibiting ACE was 21.0 ⁇ mol/L, 24.6 ⁇ mol/L and 23.8 ⁇ mol/L, respectively.
  • the present invention isolated a new Ganoderma lucidum strain SCIM 1006 capable of producing new antihypertensive peptides, which was identified as Chinese Ganoderma lingzhi (Ganoderma lingzhi), and the produced antihypertensive peptides were structured and functioned Verification: Compared with the previously reported ACE inhibitory peptides, the Ganoderma mycelium antihypertensive peptide Ser-Tyr-Pro obtained by the present invention not only has stronger ACE inhibitory activity, IC50 is 62.5 ⁇ g/mL, and its molecular weight is only It is 366Da, which is easily absorbed by the human body when it enters the intestinal tract.
  • a method for separating and preparing high-efficiency antihypertensive peptides from Chinese Ganoderma lucidum mycelium was obtained.
  • Ganoderma lucidum mycelium was added to stimulate peptide production by adding 17 kinds of mixed amino acids, and 3kDa membrane ultrafiltration was used to prepare pure natural, low-molecular-weight, highly active Ganoderma lucidum mycelium antihypertensive peptide.
  • the pressure peptide is highly purified to prepare pure natural, low molecular weight, high activity, and high purity pure Ganoderma lucidum mycelium antihypertensive peptide.
  • the preparation method has strong operability, short production cycle and easy large-scale production.
  • the first object of the present invention is to provide a peptide whose amino acid sequence is Ser-Tyr-Pro (as shown in SEQ ID NO: 1).
  • the peptide is a hypotensive peptide, which belongs to a competitive inhibitory peptide that inhibits ACE activity.
  • Ser and Lys472 in the ACE active area form a salt bond
  • Ser and Lys472 in the ACE active area Tyr and Gln242
  • Pro and Lys415 form three hydrogen bonds
  • Tyr, Pro and Tyr484 and Phe488 in the ACE active area form three hydrogen bonds. It acts to occupy the active area of ACE, making it unable to act as a substrate and inactivate.
  • the peptide can significantly reduce arterial diastolic blood pressure through intraperitoneal injection and intravenous injection of the antihypertensive peptide; in the body, by up-regulating the eNOS phosphorylation level of vascular endothelial cells induced by angiotensin I, the expression of endothelin-1 is inhibited, Play a blood pressure lowering activity.
  • the second objective of the present invention is to provide a Ganoderma strain SCIM 1006 (Ganoderma lingzhi), which was deposited in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection and Management Committee on November 25, 2019, and the deposit number is CGMCC No .18819, the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • CGMCC General Microbiology Center
  • the Ganoderma lucidum strain SCIM 1006 can produce the antihypertensive peptide whose amino acid sequence is Ser-Tyr-Pro.
  • the third object of the present invention is to provide a method for producing the peptide.
  • the method uses artificial synthesis, biological fermentation, or enzymatic hydrolysis for production.
  • the method includes using the Ganoderma lucidum strain SCIM 1006 of the present invention for fermentation production.
  • the culture medium used in the fermentation contains ingredients required for normal cultivation of Ganoderma lucidum.
  • the culture medium used in the fermentation contains 0.5-0.6% NH 4 Cl, 4.1-4.2% soluble starch, 0.005% vitamin B1, and 0.15% K 2 HPO 4 .
  • the fermentation production further includes using a fermentation medium containing mixed amino acids.
  • the mixed amino acids in the fermentation medium include the following two or more amino acids: Ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser, Gly, Arg, Ala.
  • the mixed amino acid in the fermentation medium is a mixture of the following 17 amino acids: Ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser, Gly, Arg, Ala.
  • the added amount of a single amino acid in the fermentation medium is 0.02 g/L and above.
  • the preparation method of the fermentation medium includes: excluding amino acids, dissolving the components of the medium in water, adjusting the pH to 6.8-7.0, and sterilizing; formulating the amino acids into a high-concentration mother liquor and pressing The final concentration of 0.02g/L or more is added to the sterilized solution after filtration with a 0.22 ⁇ m filter membrane in a sterile environment.
  • the fermentation production is to insert the mycelial seed liquid of the Ganoderma lucidum strain SCIM 1006 into a fermentation medium with an inoculum amount of 10-15% for fermentation.
  • the fermentation is performed at a temperature of 20-28° C. and 100-140 r/min for 6-8 days.
  • the fermentation is carried out at a temperature of 24° C. and a low-temperature and low-speed condition of 120 r/min.
  • the fermentation production is specifically:
  • Chinese Ganoderma lucidum strain SCIM 1006 (Ganoderma lingzhi) was inoculated into a seed medium with glucose as carbon source, peptone and yeast extract as nitrogen source, and KH 2 PO 4 and MgSO 4 as mineral elements.
  • the composition of the seed medium It is: glucose 4 ⁇ 4.6%, peptone 0.3 ⁇ 0.5%, yeast extract 0.2 ⁇ 0.3%, KH 2 PO 4 0.15%, MgSO 4 0.05%; then at a temperature of 28 ⁇ 30°C and a rotation speed of 150 ⁇ 180r/min Fermented in shake flasks for 7-9 days to obtain Ganoderma lucidum seed liquid;
  • the Ganoderma lucidum seed liquid into an inoculum with a cell concentration of 1.7 ⁇ 2.0g/L, and inoculate it into the liquid fermentation medium at a volume ratio of 12 ⁇ 15% at a temperature of 27 ⁇ 30°C and a rotation speed of Submerged fermentation at 160 ⁇ 190r/min for 6 ⁇ 8 days; among them, the liquid fermentation medium contains 3.9 ⁇ 4.3% soluble starch, 0.5 ⁇ 0.7% NH 4 Cl, 0.005% vitamin B1, 0.15% K 2 HPO 4 , and mixed Amino acids.
  • the fourth objective of the present invention is to provide a method for extracting and purifying the peptide from the fermentation broth of Ganoderma lucidum.
  • the Ganoderma lucidum fermentation broth is the fermentation broth of Ganoderma lucidum strain SCIM 1006.
  • the method includes: extracting and separating the antihypertensive peptide from the Ganoderma lucidum mycelium with a homogenization buffer and an ultrafiltration membrane; purifying by high performance liquid chromatography to obtain a pure product of the Ganoderma lucidum mycelium antihypertensive peptide.
  • the extraction take the Ganoderma lucidum fermentation broth, collect the hyphae, pour it into liquid nitrogen and grind to a fine granular shape, then transfer to the pre-cooled homogenization buffer, homogenize the tissue on an ice bath, and then centrifuge to obtain the supernatant; from the supernatant Collect the ultrafiltrate with a molecular weight of less than 3kDa in the liquid, and then remove the homogenization buffer in the filtrate to obtain the crude Ganoderma lucidum mycelium antihypertensive peptide.
  • the homogenization buffer is 10 mM Tris-HCl buffer, pH 7.0, containing 2 mM EDTA, 1% enzyme inhibitor Roche, 10 mM ⁇ -mercaptoethanol, and 5-10% glycerol; Place in the refrigerator at °C for 2h.
  • the homogenization is stopped for 5 seconds every 15 seconds to prevent degradation of the target peptide as much as possible.
  • the purification includes: dissolving the crude Ganoderma lucidum mycelium antihypertensive peptide in a 10% acetonitrile aqueous solution, loading the sample on a C18 solid phase extraction column, washing with a 10% acetonitrile aqueous solution, and then using 70% The acetonitrile aqueous solution was eluted, and the eluted adsorbed fraction was collected, concentrated under vacuum at low temperature to remove acetonitrile, and applied to RP-HPLC for further purification.
  • the chromatographic conditions of the RP-HPLC are: chromatographic column Znertsil ODS-3 (4.6 ⁇ 250mm), flow rate 1.0mL/min, detection wavelength 220nm, mobile phase is acetonitrile and ultrapure water, 0- 3min, 15% acetonitrile; 3-51min, 15-55% acetonitrile. Collect the components with a retention time of 8-9 minutes and a purity of more than 95%, which is the pure product of Ganoderma lucidum mycelium antihypertensive peptide.
  • the fifth object of the present invention is to provide a medicine with a blood pressure lowering effect, which contains the peptide of the present invention whose amino acid sequence is Ser-Tyr-Pro or the Ganoderma strain SCIM 1006 of the present invention.
  • the peptide with the amino acid sequence of Ser-Tyr-Pro or the Ganoderma strain SCIM 1006 of the present invention is used as the main active ingredient in the medicine.
  • the drug is a drug that has the effect of lowering arterial diastolic blood pressure.
  • the medicament also contains auxiliary materials required for preparing the medicament.
  • the drug can be a pharmaceutical composition, which can be prepared into clinically commonly used capsules, tablets, granules, pills, powders or oral liquids according to conventional preparation techniques.
  • the sixth object of the present invention is to provide a non-disease diagnosis and treatment method for regulating the expression of eNOS and endothelin-1.
  • the method comprises using a peptide with an amino acid sequence of Ser-Tyr-Pro or a peptide with an amino acid sequence of Ser -A strain of Tyr-Pro.
  • the method is to use Ser-Tyr-Pro to up-regulate the phosphorylation level of eNOS, or to inhibit the expression level of endothelin-1 protein.
  • the present invention also requires protection of nucleotide sequences encoding Ser-Tyr-Pro peptides, vectors or recombinant bacteria expressing Ser-Tyr-Pro peptides, and Ganoderma lucidum containing Ser-Tyr-Pro peptides or capable of producing Ser-Tyr-Pro peptides Any kit of strain SCIM 1006.
  • the antihypertensive peptide of the present invention has the following advantages: 1) The antihypertensive peptide has good in vivo and in vitro antihypertensive activity, and exerts antihypertensive effect by blocking the ACE-Ang I-AT1R pathway. It is effective in reducing arterial systolic blood pressure and systolic blood pressure, among which the effect of reducing diastolic blood pressure is better; 2) The antihypertensive peptide has the characteristics of low molecular weight and easy absorption by the human body.
  • the molecular weight of pure Ser-Tyr-Pro is only 366Da, It can be directly absorbed when entering the intestine; 3)
  • the crude and pure antihypertensive peptides can be used as health food additives or antihypertensive drugs;
  • the ganoderma strain SCIM 1006 of the present invention is a Chinese ganoderma (Ganoderma lingzhi) mycelium capable of producing the antihypertensive peptide Ser-Tyr-Pro.
  • the method for preparing antihypertensive peptides by fermentation of the mycelium of Ganoderma lucidum SCIM 1006 of the present invention has the following advantages compared with the existing methods: 1) The fermentation technology is highly pertinent, specifically for the modern fermentation technology of Ganoderma lucidum mycelium antihypertensive peptides , Instead of the traditional Ganoderma lucidum mycelium fermentation technology; 2) By adding exogenous amino acids to the liquid fermentation medium and combining with low-temperature and low-speed fermentation technology, the yield of natural peptides from Ganoderma lucidum mycelium is increased to 1.52g/L, much higher than The yield of traditional fermentation technology without adding exogenous amino acids.
  • the method for extracting and purifying antihypertensive peptides of the present invention prevents endogenousness by adding EDTA, enzyme inhibitor Roche, ⁇ -mercaptoethanol, glycerol and other reagents to the homogenization buffer in the process of extracting and separating antihypertensive peptides from Ganoderma lucidum mycelium. Protease degrades the target peptide to ensure the quality of the product.
  • the proportion of peptides with a molecular weight of less than 2kDa in the obtained crude product accounts for 86.8%; solid phase extraction column and RP-HPLC two-step chromatography are used to purify Ganoderma lucidum mycelium antihypertensive peptide with purity Up to 95%, simple operation, fast and convenient, easy to industrialized production.
  • CGMCC General Microbiology Center
  • the deposit number is CGMCC No.18819, and the deposit address is Beijing No. 3, No. 1, Beichen West Road, Chaoyang District.
  • Figure 1 shows the sequence of Ganoderma lucidum strain SCIM 1006ITS.
  • Figure 2 is the molecular phylogenetic tree of Ganoderma lucidum strain SCIM 1006.
  • Figure 3 is the RP-HPLC chromatogram of Ganoderma lucidum mycelium antihypertensive peptide; collected components (a); ACE inhibition rate determination (b).
  • Figure 4 is the ACE inhibitory mechanism of Ganoderma lucidum mycelium antihypertensive peptide; among them,
  • Figure 5 is the Ganoderma lucidum mycelium antihypertensive peptide gastrointestinal simulated digestion analysis.
  • the antihypertensive peptide (1mg/mL) was digested at 37°C for 30 minutes in a digestion solution containing pepsin (0.5%, w/w) and pH 2.0, and then continued to use NaOH to adjust the pH of the pepsin digestion solution to 7.5 , Add pancreatin (0.5%, w/w) and digest at 37°C for 90 min.
  • Figure 6 is the evaluation of the antihypertensive effect of Ganoderma lucidum mycelium antihypertensive peptide in vivo; among them,
  • Figure 7 is the analysis of the antihypertensive activity of Ganoderma lucidum mycelium in vitro; among them,
  • a 1 is the measured absorbance of the control group
  • a 2 is the measured absorbance of the experimental group
  • a 0 is the measured absorbance of the blank group
  • IC 50 represents the concentration of the sample corresponding to the ACE inhibition rate of 50%, that is , the smaller the IC 50 value, the stronger the ACE inhibitory activity of the sample.
  • the lyophilized sample was dissolved in an aqueous solution containing 1% acetic acid and 50% methanol, and the LTQ Velos Pro mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect Ganoderma lucidum SCIM 1006 hyphae with HESI-II ESI probe
  • the amino acid sequence of the antihypertensive peptide in the purified fraction of the body was identified by mass spectrometry.
  • Use LTQ Tune software to operate sample loading, experiment settings and data collection, and use Xcalibur software to analyze the data.
  • the mass spectrometry conditions are set as follows: the syringe pump is set to offline sample loading mode, the flow rate is 2.5 ⁇ 15.0 ⁇ L/min; the heat source heater and capillary temperature are 60°C and 300°C respectively; sheath gas flow with aux/scavenger It is 5 ⁇ 10arb; it adopts the positive detection method, and the spray voltage is 3.5kV.
  • the target peptide ion is subjected to collision-induced dissociation, the collision energy is 28-35%, the activation Q value is 0.25, and the activation time is 10 ms.
  • the amino acid sequence of the target peptide is mainly analyzed by following the b-type cleavage pathway and cross-validated with all the main product ions obtained.
  • each hyphae was ground in liquid nitrogen in a mortar and used for genomic DNA extraction, followed by adding 600 ⁇ L TE, 250 ⁇ L 10% SDS, 3 ⁇ L 20 ng/ ⁇ L proteinase K, and bathing in 37°C water for 1 hour. Then add 150 ⁇ L of 5mol/L NaCl, and then 150 ⁇ L of 2% CTAB, and incubate in a water bath at 65°C for 20 minutes. Centrifuge at high speed for 20 min, take the supernatant and add an equal volume of isopropanol, and place at room temperature for 30 min. Centrifuge at high speed for 10 minutes, add 750 ⁇ L 70% alcohol to the precipitate.
  • the molecular weight of the PCR amplified product of Ganoderma lucidum strain SCIM 1006 is 621 bp (sequence such as SEQ ID NO: 2).
  • the sequence was submitted to the GenBank nucleic acid sequence database for sequence comparison, and it was found that the strain was compared with G. lingzhi sp.
  • the sequence similarity of nov. (East Asia) is 100%, while the homology with G. multipileum (Tropical Asia) is relatively low. Therefore, the genus of Ganoderma lucidum strain SCIM 1006 was identified as G. lingzhi.
  • the ITS sequence of Ganoderma lucidum strain SCIM 1006 is as follows:
  • CGMCC General Microbiology Center
  • CGMCC China Microbial Culture Collection Management Committee
  • the deposit number is CGMCC No.18819, and the deposit address is Chaoyang, Beijing. No. 3, No. 1, Beichen West Road, District.
  • the strain used Ganoderma lucidum strain SCIM 1006, the deposit number is CGMCC No.18819.
  • the fermentation medium was prepared.
  • the medium formula was: NH 4 Cl 0.5%, 17 kinds of mixed amino acids (Ler, Trp, Val, Tyr, Asp, Phe, Glu, Asn, Thr, Gln, Cys, His, Pro, Ser , Gly, Arg, Ala, each amino acid is added at 0.02g/L, soluble starch is 4.1%, vitamin B1 is 0.005%, K 2 HPO 4 is 0.15%. Except amino acids, dissolve each component of the fermentation medium Adjust the pH to 7.0 in distilled water and autoclave at 121°C for 30 minutes.
  • the 17 kinds of mixed amino acids are prepared into a high-concentration mother liquor, which is added to the sterilized fermentation culture in a sterile ultra-clean table with a 0.22 ⁇ m filter according to the addition ratio. In the base, mix well.
  • the Chinese Ganoderma lucidum strain SCIM 1006 (Ganoderma lingzhi) was inoculated into a seed medium with glucose as a carbon source, peptone and yeast extract as a nitrogen source, and KH 2 PO 4 and MgSO 4 as mineral elements.
  • the composition of the seed medium was glucose 4.2%, peptone 0.4%, yeast extract 0.25%, KH 2 PO 4 0.15%, MgSO 4 0.05%; then fermented in a shake flask at a temperature of 28°C and a rotation speed of 150 r/min for 8 days to obtain a Ganoderma lucidum seed liquid;
  • the Ganoderma lucidum seed liquid was prepared into an inoculum with a bacterial concentration of 2.0g/L, and inoculated into the liquid fermentation medium at a volume ratio of 15%.
  • the fermentation was carried out for 7 days at a temperature of 24°C and a low temperature and a low speed of 120r/min. .
  • the yield of natural peptides from Ganoderma lucidum mycelium reached 1.52g/L.
  • the homogenization buffer is 10 mM Tris-HCl buffer, pH 7.0, containing 2 mM EDTA, 1% enzyme inhibitor Roche, 10 mM ⁇ -mercaptoethanol, 5-10% glycerol, and placed in a refrigerator at 4°C for 2 hours before use. Homogenize the Ganoderma lucidum mycelium homogenate on an ice bath for 20 minutes, and stop the operation for 5 seconds every 15 seconds to prevent the target peptide from degrading. After homogenization, it was centrifuged (4°C, 12000g) for 20 minutes, and centrifuged twice. Take the supernatant and perform ultrafiltration with 100kDa and 3kDa ultrafiltration membranes respectively.
  • the ACE inhibitory activity and molecular weight distribution ratio were measured, and it was found that the ACE inhibitory activity IC 50 was 480.0 ⁇ g/mL, the polypeptide content was 1.52 mg/mL, and the percentage of polypeptides with a molecular weight of less than 2kDa accounted for 86.8%.
  • the chromatographic conditions were Znertsil ODS-3 (4.6 ⁇ 250mm), Flow rate 1.0mL/min, detection wavelength 220nm, mobile phase is acetonitrile and ultrapure water, 0-3min, 15% acetonitrile; 3-51min, 15-55% acetonitrile; 51-53min, 55-95% acetonitrile; 53-58min , 95% acetonitrile; 58-59min, 95-15% acetonitrile; 59-60min, 15% acetonitrile. Collect a tube every 2.5 min from the mobile phase outlet, for a total of 24 tubes. The purification chromatogram is shown in Figure 3.
  • each tube Concentrate each tube with a vacuum concentrator to a peptide concentration of 100 ⁇ g/mL, determine its ACE inhibitory activity, and further use ESI-MS to identify the amino acid sequence of each tube with a molecular weight of less than 2kDa.
  • the ACE inhibitory activity and identification results of some sequences are shown in the table Shown in 1 (the amino acid sequence is shown in SEQ ID NO: 1, and SEQ ID NO: 9-SEQ ID NO: 21).
  • the small peptide with the sequence Ser-Tyr-Pro (amino acid sequence shown in SEQ ID NO: 1) has the strongest activity, with an IC 50 of 62.5 ⁇ g/mL, and the highest yield, reaching 0.67%.
  • the small peptide Ser-Tyr-Pro corresponds to a retention time of about 8.0 minutes, and its purity is 95.0% after testing.
  • the small peptide is a pure product of Ganoderma lucidum mycelium antihypertensive peptide.
  • the Lineweaver-Burk double reciprocal mapping method was used to analyze the inhibitory model of Ganoderma lucidum mycelium antihypertensive peptide.
  • the ACE was incubated with different mass concentrations (0, 50, 100 ⁇ g/mL) of antihypertensive peptide Ser-Tyr-Pro.
  • H H L substrate concentration 0.8, 1.6, 3.2, 6.4mmol/L
  • the reaction conditions are the same as the above-mentioned "ACE inhibitory activity determination”
  • the absorbance at 228nm is measured
  • the Lineweaver-Burk double reciprocal graph is drawn
  • the kinetic parameters are calculated, Maximum initial velocity (Vmax) and Michaelis-Menten constant (Km).
  • Chromatographic conditions Chromatographic column is Znertsil ODS-3 (4.6 ⁇ 250mm); fluidity is water (containing 0.04% trifluoroacetic acid), acetonitrile (0.03% trifluoroacetic acid); elution conditions are 0-5min, 0% acetonitrile; 5-40min, 0-40% acetonitrile; temperature is 25°C; detection wavelength is 220nm; flow rate is 0.5mL/min.
  • Ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro can be degraded by pepsin but not by pancreatin, indicating that it has good stability in the intestinal tract.
  • Example 4 In vivo and in vitro blood pressure lowering efficacy analysis of antihypertensive peptide Ser-Tyr-Pro
  • the 8-week male spontaneously hypertensive rat SHR with tail artery systolic pressure exceeding 180mmHg provided by Hunan Slack Jingda Experimental Animal Co., Ltd. was selected. They were reared at a constant temperature of 23°C for 12 hours in light and 12 hours in darkness, with free intake of food and water, and adaptive rearing for 1 week. SHR was randomly divided into 3 groups, each with 6 animals. The first group was the negative control group and was injected with the same dose of normal saline, the second group was administered by intravenous injection, and the third group was administered by intraperitoneal injection.
  • Ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro was administered once at a prescribed dose of 10 mg/kg animal body weight.
  • the systolic and diastolic blood pressure of the tail artery were measured 0-8h after the administration of SHR, and the blood pressure value of each rat was measured. Take the average of 5 times.
  • intraperitoneal injection or intravenous injection of the antihypertensive peptide can reduce SHR diastolic and systolic blood pressure. After 2 hours of administration, both systolic blood pressure and diastolic blood pressure decreased significantly. Because the antihypertensive peptide was partially metabolized and degraded in the blood, blood pressure increased after 3 hours of administration. Among them, it can be seen from Figure 6a that the systolic blood pressure of the non-administered group was 160.7mmHg when the drug was administered for 2 hours.
  • intravenous injection had a better effect on reducing systolic blood pressure, and the systolic blood pressure was reduced to 122.6mmHg; In Figure 6b, the diastolic blood pressure of the non-administered group was 134.7mmHg.
  • Intravenous injection has a better blood pressure lowering effect than intraperitoneal injection, and the diastolic blood pressure can be reduced to 96.3mmHg.
  • Human umbilical cord vein endothelial cells are cultured with RPMI 1640 culture medium, which also contains 10% fetal bovine serum, 100 ⁇ g/mL penicillin and 100 ⁇ g/mL streptomycin. Microscopic observation and MTS cell viability assay were used to analyze the regulation of the antihypertensive peptide Ser-Tyr-Pro on the expression of eNOS and endothelin-1 in HUVEC induced by angiotensin I.
  • HUVEC (1 ⁇ 10 5 cells/mL) were planted in a 10 cm cell culture dish and cultured in a carbon dioxide incubator containing 5% CO 2 at 37°C. After the cells mature, remove the upper layer carefully, add 1mLhanks solution, remove the upper layer, repeat 3 times, add 4mL Trypsin-EDTA for digestion, after the cells shrink and become round, add 12mL RPMI 1640 culture solution to terminate the digestion. Divide them into 10 cm petri dishes (2 mL/dish), a total of 5 dishes, add fresh culture solution to each dish to 10 mL/dish.
  • angiotensin I (1.0 mg/mL) was added to induce 2 hours to establish a hypertensive cell model.
  • Different concentrations (0, 25, 50, 100 ⁇ g/mL) of antihypertensive peptide Ser-Tyr-Pro were added respectively, in which angiotensin I and antihypertensive peptide were not added as the control group, and the antihypertensive peptide was not added as the induction Group, extract total protein and use Wester-blot technology to detect the expression level of eNOS and endothelin-1 protein.
  • HUVEC (1 ⁇ 10 5 cells/mL) were planted in a 10 cm cell culture dish and cultured in a carbon dioxide incubator containing 5% CO 2 at 37°C. After the cells mature, remove the upper layer carefully, add 1mL hanks solution, remove the upper layer, repeat 3 times, add 4mL Trypsin-EDTA for digestion, after the cells shrink and become round, add 12mL RPMI 1640 medium to terminate the digestion. Divide them into 6-well plates (2mL/well), a total of 5 wells. After the cells were overgrown to 80%, angiotensin I (1.0 mg/mL) was added to induce 2 hours to establish a hypertensive cell model.
  • angiotensin I 1.0 mg/mL
  • Endothelin-1 anti-chain 5'CAG AAA CTC CAC CCC TGT GT3' (sequence shown in SEQ ID NO: 3),
  • Endothelin-1 positive chain 5'TCC TCT GCT GGT TCC TGA CT 3'(sequence is shown in SEQ ID NO: 4)
  • eNOS anti-chain 5’GGT GGC CCT CGT GGA CTT GC 3’ (sequence is shown in SEQ ID NO: 5)
  • eNOS positive chain 5’AGG CCT TCC GAG GCT GG 3’ (sequence is shown in SEQ ID NO: 6)
  • ⁇ -actin anti-chain 5'CAT GTA CGT TGC TAT CCA GGC 3'(sequence is shown in SEQ ID NO: 7)
  • ⁇ -actin positive chain 5'CTC CTT AAT GTC ACG CAC GAT 3'(sequence is shown in SEQ ID NO: 8).
  • the regulation of the mRNA level of eNOS and endothelin-1 by Ganoderma lucidum mycelium antihypertensive peptide Ser-Tyr-Pro is similar to the change trend of its protein expression level.
  • the mRNA level of eNOS increased by 3.51 times, and the mRNA level of endothelin-1 decreased by 27.3%.

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Abstract

本发明公开一种灵芝菌丝体降压肽及其制备方法，属于多肽生物技术及降血压药物技术领域。本发明的降压肽，具有良好的体内外降血压活性，以及分子量低、易被人体吸收的特点，可作为保健食品添加剂或降血压药物；本发明的灵芝菌株SCIM 1006，是能产降压肽Ser-Tyr-Pro的中国灵芝菌丝体，其通过在液体发酵培养基中添加外源氨基酸并联合低温低转速发酵技术，其天然多肽产量提高至1.52g/L；同时本发明的降压肽提取纯化方法，操作简单，快速方便，易于工业化生产。

Description

一种灵芝菌丝体降压肽及其制备方法 技术领域
本发明涉及一种灵芝菌丝体降压肽及其制备方法，属于多肽生物技术及降血压药物技术领域。
背景技术
高血压是威胁人类健康的主要疾病之一，是一种生活中常见的心脑血管疾病。随着现代社会的发展和人民生活水平的提高，高血压心脑血管疾病发病率大大增加，愈趋于年轻化。据《2018世界卫生统计报告》统计显示，在非传染性疾病中心脑血管疾病死亡率居首，死亡人数达1790万，而我国高血压患者已累计超过3亿人。然而，公认的卡托普利、雷米普利、赖诺普利等降血压合成药物，在临床应用过程中存在疲乏恶心、剧烈咳嗽、皮疹瘙痒等副作用。因此，亟待寻求相对安全高效、无毒副作用的降血压药物。近年来，来源于食物蛋白降解产物的活性肽以其高效安全、人体易吸收等优点倍受关注。
血管紧张素转化酶(Angiotensin I converting enzyme，ACE)属血管内皮细胞膜结合酶，它催化血管紧张素Ⅰ转化为血管紧张素Ⅱ导致血压升高，是治疗高血压的理想靶点。ACE抑制肽是一类具有潜在降血压功效的小分子物质，可抑制ACE酶活，降低舒缓激肽的破坏和减少血管紧张素I的降解。国内外相继从动物制品、海洋生物、植物(Seung,et al.Antihypertensive peptides from animal products,marine organisms,and plans-a review.Food chemistry,2017,228:506-517)中获得ACE抑制肽，虽在体外具有很强的ACE抑制活性，但并不能说明其能在体内发挥降血压功效，且ACE抑制肽的降血压分子机制并不清楚。此外，现在普遍采用定向酶解技术，对食物来源蛋白进行降解以制备ACE抑制肽。然而，定向酶解技术所获得的酶解产物成分相对复杂，后续纯化困难；酶解过程需严格控制蛋白酶的酶解条件(加酶量、温度及pH等)，将消耗更多“物力+能源+人力”，生产成本大，因而制约了ACE抑制肽规模化生产的发展。目前，国外少数研究者已从传统发酵产品中发现天然的ACE抑制肽，开辟了利用微生物制备天然ACE抑制肽的新思路。
目前报道的ACE抑制肽，包括墨鱼肌肉蛋白Val-Glu-Leu-Tyr-Pro、鸡蛋His-Leu-Phe-Gly-Pro-Pro-Gly-Lys-Lys-Asp-Pro-Val、蚕蛹Gly-Ala-Met-Val-Val-His、花生Tyr-Leu-Val-Arg等不同来源的肽类物质，分子量大小不一，通常由4-12氨基酸残基组成，虽在体外具有较强的ACE抑制活性，但大部分研究者并未对其体内降血压功效进行验证，体内降血压机制尚不明确。
灵芝属真菌，药理价值极高，具有抗肿瘤、调节免疫、保肝降压等多种功效。三萜类化合物和多糖是灵芝的两大主要活性成分，而有关灵芝活性寡肽的报道甚少。灵芝子实体栽培周期较长(4～9个月)，易受季节与环境的影响，因而子实体活性物质生物稳定性较差，而通过分离灵芝细胞并利用液态发酵技术，制备灵芝菌丝体来获取活性物质是更为有效的手段。因此，将灵芝菌丝体作为一种制备活性肽的新资源进行开发利用具有重要的现实意义。在前期研究过程中，发明人团队从灵芝菌丝体中获得了3种ACE抑制肽，其氨基酸序列分别为Gln-Leu-Asp-Leu、Gln-Leu-Val-Pro和Gln-Asp-Val-Leu，测得其抑制ACE的IC50分别为21.0μmol/L、24.6μmol/L和23.8μmol/L。但是，仍然很有必要开发更多的具有ACE抑制的肽，以及活性更好的ACE抑制肽。
发明内容
为了解决上述至少一个技术问题，本发明分离到一株能够产新型降压肽的灵芝新菌株SCIM 1006，经鉴定为中国灵芝(Ganoderma lingzhi)，并对其产生的降压肽进行了结构和功能验证；相较于现有报道的这些ACE抑制肽，本发明获得的灵芝菌丝体降压肽Ser-Tyr-Pro，不仅具有较强的ACE抑制活性，IC50为62.5μg/mL，其分子量仅为366Da,进入肠道易被人体吸收；同时，具有良好的体内降血压功效，通过阻断ACE-Ang I-AT1R通路发挥降血压功效，对降低动脉收缩压和收缩压均有效果。
在此基础上，经过大量摸索实验，获得一种从中国灵芝菌丝体中分离、制备高效降压多肽的方法。通过添加17种混合氨基酸刺激灵芝菌丝体产生多肽，并采用3kDa膜超滤制得纯天然、低分子量、高活性的灵芝菌丝体降压肽粗品，最后利用高效液相色谱技术对该降压肽进行高度纯化，制备出纯天然、低分子量、高活性、高纯度的灵芝菌丝体降压肽纯品，该制备方法可操作性强、生产周期短、易规模化生产。
本发明的第一个目的是提供一种肽，所述肽的氨基酸序列为Ser-Tyr-Pro(如SEQ ID NO:1所示)。
所述肽为降压肽，属于一种抑制ACE活性的竞争性抑制肽。通过Ser与ACE活性区域的Lys472形成一个盐键，通过Ser与ACE活性区域的Lys472、Tyr与Gln242、Pro与Lys415形成三个氢键，并通过Tyr、Pro与ACE活性区域的Tyr484、Phe488产生疏水作用，从而占据ACE的活性区域，使其不能作用底物而失活。
所述肽，通过腹腔注射及静脉注射该降压肽，均可显著降低动脉舒张压；在机体内通过上调由血管紧张素I诱导血管内皮细胞的eNOS磷酸化水平，抑制内皮素-1表达，发挥降血压活性。
本发明的第二个目的是提供一株灵芝菌株SCIM 1006(Ganoderma lingzhi)，该菌株于2019年11月25日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.18819，保藏地址为北京市朝阳区北辰西路1号院3号。
所述灵芝菌株SCIM 1006，能够生产氨基酸序列为Ser-Tyr-Pro的降压肽。
本发明的第三个目的是提供生产所述肽的方法。
在一种实施方式中，所述方法是采用人工合成法或者生物发酵法或者酶解法进行生产。
在一种实施方式中，所述方法包括利用本发明的灵芝菌株SCIM 1006进行发酵生产。
在一种实施方式中，所述发酵使用的培养基中，含有正常培养灵芝所需要的成分。比如，含有0.5～0.6％NH 4Cl，4.1～4.2％可溶性淀粉，0.005％维生素B1，0.15％K 2HPO 4。
在一种实施方式中，所述发酵生产，还包括利用含有混合氨基酸的发酵培养基。
在一种实施方式中，所述发酵培养基中的混合氨基酸包括如下两种以上的氨基酸：Ler、Trp、Val、Tyr、Asp、Phe、Glu、Asn、Thr、Gln、Cys、His、Pro、Ser、Gly、Arg、Ala。
在一种实施方式中，所述发酵培养基中的混合氨基酸为如下17种氨基酸的混合：Ler、Trp、Val、Tyr、Asp、Phe、Glu、Asn、Thr、Gln、Cys、His、Pro、Ser、Gly、Arg、Ala。
在一种实施方式中，所述发酵培养基中，单种氨基酸的添加量为0.02g/L及以上。
在一种实施方式中，所述发酵培养基的制备方法包括：除氨基酸以外，将培养基各成分溶于水中，将pH调至6.8～7.0，灭菌；将氨基酸配制成高浓度母液，按终浓度为0.02g/L以上的添加量在无菌环境中以0.22μm滤膜过滤，加入已灭菌的溶液中。
在一种实施方式中，所述发酵生产，是将灵芝菌株SCIM 1006的菌丝体种子液以10～15％接种量接入发酵培养基中，进行发酵。
在一种实施方式中，所述发酵，是在温度20-28℃、100-140r/min的条件下，发酵6～8d。
在一种实施方式中，所述发酵，是在温度24℃、120r/min的低温低转速条件下进行。
在一种实施方式中，所述发酵生产，具体是：
(1)将中国灵芝菌种SCIM 1006(Ganoderma lingzhi)接种到以葡萄糖为碳源、蛋白胨和酵母膏为氮源、KH 2PO 4和MgSO 4为矿物元素的种子培养基中，种子培养基组成为：葡萄糖4～4.6％，蛋白胨0.3～0.5％，酵母膏0.2～0.3％，KH 2PO 40.15％，MgSO 40.05％；然后在温度为28～30℃、转速为150～180r/min的摇瓶中发酵7～9天，获得灵芝种子液；
(2)将灵芝种子液制备成菌体浓度为1.7～2.0g/L的接种液，按体积比12～15％的比例接种到液体发酵培养基中，在温度为27～30℃、转速为160～190r/min下深层发酵6～8天；其中，液体发酵培养基中含有3.9～4.3％可溶性淀粉，0.5～0.7％NH 4Cl，0.005％维生素B1， 0.15％K 2HPO 4，以及混合氨基酸。
本发明的第四个目的是提供从灵芝发酵液中提取纯化所述肽的方法。所述灵芝发酵液为灵芝菌株SCIM 1006的发酵液。
所述方法，包括：用匀浆缓冲液和超滤膜对灵芝菌丝体中降压肽进行提取分离；通过高效液相色谱纯化，获得灵芝菌丝体降压肽纯品。
所述提取：取灵芝发酵液，收集菌丝，倒入液氮研磨至细粒状，然后转移至预冷匀浆缓冲液中，于冰浴上组织匀浆，然后离心取上清；从上清液中收集分子量小于3kDa超滤液，然后去除滤液中的匀浆缓冲液，即得到灵芝菌丝体降压肽粗品。
在一种实施方式中，所述匀浆缓冲液为10mM Tris-HCl缓冲液，pH 7.0，含2mM EDTA、1％酶抑制剂Roche、10mMβ-巯基乙醇、5～10％甘油；用前在4℃冰箱中放置2h。
在一种实施方式中，所述组织匀浆时，每匀浆15s则停5s，尽可能防止目标肽降解。
所述纯化：将灵芝菌丝体降压肽粗品溶于乙腈水溶液中，上样于C18固相萃取柱，经流洗和洗脱后，收集洗脱下来的吸附部分，去除乙腈，然后上样于RP-HPLC作进一步纯化。
在一种实施方式中，所述纯化包括：将灵芝菌丝体降压肽粗品溶于10％乙腈水溶液中，上样于C18固相萃取柱，用10％乙腈水溶液流洗，再用70％乙腈水溶液洗脱，收集洗脱下来的吸附部分，真空低温浓缩以去除乙腈，上样于RP-HPLC作进一步纯化。
在一种实施方式中，所述RP-HPLC的色谱条件为：色谱柱Znertsil ODS-3(4.6×250mm),流速1.0mL/min，检测波长220nm，流动相为乙腈和超纯水，0-3min，15％乙腈；3-51min，15-55％乙腈。收集保留时间在8～9min且纯度达95％以上的组分，即为灵芝菌丝体降压肽纯品。
本发明的第五个目的是提供一种具有降血压作用的药物，所述药物中含有本发明的氨基酸序列为Ser-Tyr-Pro的肽或者本发明的灵芝菌株SCIM 1006。
在一种实施方式中，所述药物中以氨基酸序列为Ser-Tyr-Pro的肽或者本发明的灵芝菌株SCIM 1006作为主要有效成分。
在一种实施方式中，所述药物为具有降低动脉舒张压效果的药物。
在一种实施方式中，所述药物中还含有制备药物所需的辅料。
在一种实施方式中，所述药物可以是药物组合物，可按常规制剂工艺制备成临床常用的胶囊剂、片剂、颗粒剂、丸剂、散剂或口服液。
本发明的第六个目的是提供一种非疾病的诊断和治疗的调控eNOS和内皮素-1表达的方法，所述方法包括利用氨基酸序列为Ser-Tyr-Pro的肽或者产氨基酸序列为Ser-Tyr-Pro的菌株。
在一种实施方式中，所述方法是，利用Ser-Tyr-Pro上调eNOS磷酸化水平，或者抑制内皮素-1蛋白表达水平。
本发明还要求保护编码Ser-Tyr-Pro肽的核苷酸序列、表达Ser-Tyr-Pro肽的载体或重组菌，以及含有Ser-Tyr-Pro肽或能生产Ser-Tyr-Pro肽的灵芝菌株SCIM 1006的任意的试剂盒。
本发明的有益效果：
本发明的一种降压肽，与现有活性肽相比具有以下优势：1)该降压肽具有良好的体内外降血压活性，通过阻断ACE-Ang I-AT1R通路发挥降血压功效，对降低动脉收缩压和收缩压均有效果，其中对降低舒张压效果更佳；2)该降压肽具有分子量低、易被人体吸收的特点，纯品Ser-Tyr-Pro分子量仅为366Da，进入肠道内可直接被吸收；3)该降压肽粗品和纯品可作为保健食品添加剂或降血压药物；
本发明的灵芝菌株SCIM 1006，是能产降压肽Ser-Tyr-Pro的中国灵芝(Ganoderma lingzhi)菌丝体。
本发明的利用灵芝SCIM 1006的菌丝体发酵制备降压肽方法，与现有方法相比具有以下优势：1)该发酵技术针对性强，具体针对灵芝菌丝体降压肽的现代发酵技术，而非传统的灵芝菌丝体发酵技术；2)通过在液体发酵培养基中添加外源氨基酸并联合低温低转速发酵技术，灵芝菌丝体天然多肽产量提高至1.52g/L，远高于未添加外源氨基酸的传统发酵技术产量。
本发明的降压肽提取纯化方法，通过在灵芝菌丝体降压肽提取分离过程中的匀浆缓冲液中添加EDTA、酶抑制剂Roche、β-巯基乙醇、甘油等试剂，防止内源性蛋白酶降解目标肽，保证了产品质量，得到的粗品中分子量<2kDa以下的多肽比例占86.8％；采用固相萃取柱和RP-HPLC两步层析技术法纯化灵芝菌丝体降压肽，纯度达95％以上，操作简单，快速方便，易于工业化生产。
生物材料保藏信息
一株灵芝菌株SCIM 1006，分类命名为灵芝Ganoderma lingzhi，已于2019年11月25日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.18819，保藏地址为北京市朝阳区北辰西路1号院3号。
附图说明
图1是灵芝菌株SCIM 1006ITS序列。
图2是灵芝菌株SCIM 1006的分子系统发育树。
图3是灵芝菌丝体降压肽的RP-HPLC色谱图；收集组分(a)；ACE抑制率测定(b)。
图4是灵芝菌丝体降压肽的ACE抑制机理；其中，
(a)该降压肽的竞争性抑制模型(100μg/mL(●)，50μg/mL(○)和0μg/mL(▲))；(b)该降压肽的稳态仿真分析(左上角图：势能分析的能量最小化；右上角图：温度分析NVT；左下角图：密度；右下角图：NPT的降压肽-ACE复合体复杂性和蛋白骨架RMSD分析)；(c)降压肽与ACE结合的分子动力学模拟。
图5是灵芝菌丝体降压肽胃肠模拟消化分析。该降压肽(1mg/mL)在含有胃蛋白酶(0.5％,w/w)、pH为2.0消化液中的状态，于37℃消化30min，再继续用NaOH将胃蛋白酶消化液pH调至7.5，加入胰酶(0.5％,w/w)于37℃消化90min。
图6是灵芝菌丝体降压肽体内降血压功效评价；其中，
(a)给药前后动脉收缩压变化；(b)给药前后动脉舒张压变化。以10mg/kg body weight剂量进行腹腔注射(○),静脉注射(●)和对照组(△)，给药后0～8h测定动脉血压。
图7是灵芝菌丝体体外降血压活性分析；其中，
(a)eNOS的mRNA表达水平；(b)内皮素-1的mRNA表达水平；(c)磷酸化p-eNOS蛋白表达水平；(d)内皮素-1蛋白表达水平；(e)利用Wester-blot技术分析p-eNOS和内皮素-1蛋白表达情况。
具体实施方式
1、ACE抑制活性测定方法：
往500μL离心管中加入125μL底物HHL溶液(100mmol/L硼酸盐缓冲液，pH8.3，含0.3mol/L氯化钠)，再次加入50μL样品于37℃预热5min，加入50μL ACE溶液启动反应，在37℃水浴中反应60min。反应结束后，加入125μL的1mol/L盐酸终止反应，继续加入750μL乙酸乙酯，充分混匀。1000g离心5min后，吸取500μL上层乙酸乙酯抽提液于干净的离心管中，采用真空浓缩装置挥发掉乙酸乙酯，加入1.5mL去离子水将抽提的马尿酸充分溶解，于228nm紫外波长下测定吸光值。其中，对照组用缓冲液代替样品，空白组则用缓冲液代替ACE。ACE抑制率的计算如下：
ACE抑制率(％)＝(A 1–A 2)/(A 1-A 0)×100
式中：A 1为对照组测得吸光值；
A 2为实验组测得吸光值；
A 0为空白组测得吸光值；
IC 50表示ACE抑制率为50％时所对应的样品浓度，即IC 50值越小，该样品ACE抑制活性越强。配制不同浓度的样品溶液，以样品浓度为横坐标，以ACE抑制率为纵坐标，绘制圆滑曲线，根据曲线计算IC 50。
2、ESI-MS鉴定方法：
将冻干的样品溶解在含1％乙酸和50％甲醇的水溶液中，使用LTQ Velos Pro质谱仪(Thermo Fisher Scientific,Waltham,MA,USA)，通过HESI-II ESI探针对灵芝SCIM 1006菌丝体纯化组分中降压肽的氨基酸序列进行质谱鉴定。利用LTQ Tune软件操作进行样品上样、实验设置和数据收集，并使用Xcalibur软件对数据进行分析。质谱条件设置为：将注射泵设置为离线上样模式，流速为,2.5～15.0μL/min；热源加热器和毛细管温度分别为60℃和300℃；带aux/扫气器的护套气体流量为5～10arb；采用正检测方式，喷淋电压为3.5kV。对目标肽离子进行碰撞诱导解离，碰撞能量为28～35％，激活Q值为0.25，激活时间为10ms。目标肽的氨基酸序列主要通过跟踪b型裂解途径进行解析，并与获得的所有主要产物离子进行交叉验证。
下面给出的实施例拟对本发明作进一步说明，但不能理解为是对本发明保护范围的限制，该领域的技术人员根据上述本发明的内容对本发明作出的一些非本质的改进和调整，仍属于本发明的保护范围。
实施例1：灵芝菌株的筛选和功能肽的发现
1、灵芝菌株的筛选
在湖南省长沙市岳麓山深山中灌木、落叶或杂草等受人类活动影响较小且腐殖质较多的地方，寻找形态疑似灵芝的真菌子实体，用无菌密封袋装好，立即带回实验室。将所采集的真菌子实体用清水冲洗干净，去除泥沙和污物，用纱布擦干水分，擦拭75％酒精进行表面消毒，再用解剖刀将子实体菇柄中部纵切撕开，挑取菌盖和菇柄交界处的一小块组织，转接至PDA斜面培养基上，塞好棉塞，于25℃静置培养，待菌丝铺满整个斜面时，取各菌丝0.5g于研钵中液氮研磨，加入4倍体积(v/w)纯水组织匀浆，离心(4℃，12000g)20min，取上清分别测定ACE抑制活性和多肽浓度。其中，灵芝菌株SCIM 1006，测得其水提取液的ACE抑制率为46.85％，多肽浓度为1.52mg/mL。
此外，取各菌丝0.5g于研钵中液氮研磨，用于基因组DNA提取，依次加入600μL TE、250μL 10％SDS、3μL 20ng/μL蛋白酶K，于37℃水浴1h。随后加入150μL 5mol/LNaCl，再150μL 2％CTAB，于65℃水浴20min。高速离心20min，取上清加入等体积异丙醇，室温放置30min。高速离心10min，取沉淀加入750μL 70％酒精。高速离心2min，取沉淀加入30μL纯水，4℃溶解过夜。采用ITS通用引物对各菌株DNA进行PCR扩增，并将扩增产物送至北京华大基因公司测序，在GenBank核酸序列数据库中进行同源序列比对(BLAST)，以确定其种属。
由图1可知，灵芝菌株SCIM 1006的PCR扩增产物分子量为621bp(序列如SEQ ID NO:2)，将该序列提交至GenBank核酸序列数据库进行序列比对，发现该菌株与G.lingzhi sp.nov.(East Asia)序列相似性达100％，而与G.multipileum(Tropical Asia)等菌种同源性相对较低。因此，将灵芝菌株SCIM 1006种属确定为G.lingzhi。
灵芝菌株SCIM 1006的ITS序列，具体如下：
Figure PCTCN2020072454-appb-000001
灵芝菌株SCIM 1006，分类命名为灵芝Ganoderma lingzhi，已于2019年11月25日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.18819，保藏地址为北京市朝阳区北辰西路1号院3号。
2、灵芝菌丝体的发酵培养
使用菌株：灵芝菌株SCIM 1006，保藏编号为CGMCC No.18819。
配制发酵培养基，该培养基配方为：NH 4Cl为0.5％，17种混合氨基酸(Ler、Trp、Val、Tyr、Asp、Phe、Glu、Asn、Thr、Gln、Cys、His、Pro、Ser、Gly、Arg、Ala，每种氨基酸添加量为0.02g/L，可溶性淀粉为4.1％，维生素B1为0.005％，K 2HPO 4为0.15％。除氨基酸以外，将发酵培养基各成分溶于蒸馏水中，将pH调至7.0，在121℃高压灭菌30min。17种混合氨基酸则配制成高浓度母液，按添加比例在无菌超净台中以0.22μm滤膜过加入已灭菌的发酵培养基中，充分混匀。
将中国灵芝菌种SCIM 1006(Ganoderma lingzhi)接种到以葡萄糖为碳源、蛋白胨和酵母膏为氮源、KH 2PO 4和MgSO 4为矿物元素的种子培养基中，种子培养基组成为：葡萄糖4.2％，蛋白胨0.4％，酵母膏0.25％，KH 2PO 40.15％，MgSO 40.05％；然后在温度为28℃、转速为 150r/min的摇瓶中发酵8天，获得灵芝种子液；
将灵芝种子液制备成菌体浓度为2.0g/L的接种液，按体积比15％的比例接种到液体发酵培养基中，在温度24℃、120r/min的低温低转速条件下，发酵7d。通过在液体发酵培养基中添加外源氨基酸并联合低温低转速发酵技术，灵芝菌丝体天然多肽产量达1.52g/L。
3、灵芝菌丝体降压肽的提取
取灵芝发酵培养液，用8层纱布滤去上层发酵液，用蒸馏水淋洗菌丝球4次，收集菌丝球，用滤纸充分吸去其表面水分，放入烧杯中并称重，计算灵芝菌丝体生物量。将其转移至研钵中，倒入2倍体积(v/w)液氮，研磨至细粒状，再迅速转移至4倍体积(v/w)的预冷匀浆缓冲液中。匀浆缓冲液为10mM Tris-HCl缓冲液，pH 7.0，含2mM EDTA、1％酶抑制剂Roche、10mMβ-巯基乙醇、5～10％甘油，用前在4℃冰箱中放置2h。将灵芝菌丝体匀浆液于冰浴上组织匀浆20min，该操作每匀浆15s则停5s，尽可能防止目标肽降解。匀浆后离心(4℃，12000g)20min，离心两次。取上清液，分别用100kDa和3kDa超滤膜进行超滤，收集分子量小于3kDa超滤液，用脱盐柱将超滤液中含有的匀浆缓冲液基质置换成蒸馏水，以去除2mM EDTA、1％酶抑制剂Roche、10mM β-巯基乙醇、5％甘油，所得置换液即为灵芝菌丝体降压肽粗品。测定ACE抑制活性及分子量分布比例，发现其ACE抑制活性IC 50为480.0μg/mL，多肽含量为1.52mg/mL，分子量<2kDa以下的多肽比例占86.8％。
4、灵芝菌丝体降压肽的提纯
将灵芝菌丝体降压肽粗品溶于10％乙腈水溶液中，配成浓度为100mg/mL，上样于C18固相萃取柱，用10％乙腈水溶液流洗2个柱体积，再用70％乙腈水溶液洗脱2个柱体积，收集洗脱下来的吸附部分，真空低温浓缩以去除乙腈，上样于RP-HPLC作进一步纯化，色谱条件为：色谱柱Znertsil ODS-3(4.6×250mm),流速1.0mL/min，检测波长220nm，流动相为乙腈和超纯水，0-3min，15％乙腈；3-51min，15-55％乙腈；51-53min，55-95％乙腈；53-58min，95％乙腈；58-59min，95-15％乙腈；59-60min，15％乙腈。从流动相出口以每隔2.5min收集一管，共收集24管。该纯化色谱图如图3所示。将每管用真空浓缩设备浓缩至多肽浓度为100μg/mL，测定其ACE抑制活性，并进一步用ESI-MS鉴定各管中分子量<2kDa的多肽氨基酸序列，部分序列的ACE抑制活性及鉴定结果如表1所示(氨基酸序列如SEQ ID NO:1，以及SEQ ID NO:9-SEQ ID NO:21所示)。其中，序列为Ser-Tyr-Pro(氨基酸序列如SEQ ID NO:1所示)的小肽活性最强，IC 50达62.5μg/mL，且得率最高，达0.67％。小肽Ser-Tyr-Pro所对应的保留时间约为8.0min，经检测其纯度达95.0％，该小肽即为灵芝菌丝体降压肽纯品。
表1 灵芝菌丝体小分子肽氨基酸序列的ESI-MS鉴定表及ACE抑制率
Figure PCTCN2020072454-appb-000002
实施例2：灵芝菌丝体降压肽的ACE抑制机理分析
利用Lineweaver-Burk双倒数作图法对灵芝菌丝体降压肽的抑制模型进行分析，将ACE与不同质量浓度(0、50、100μg/mL)的降压肽Ser-Tyr-Pro孵育和不同H H L底物浓度下(0.8、1.6、3.2、6.4mmol/L)混合，反应条件同上述“ACE抑制活性测定”，测定228nm吸光值，绘制Lineweaver-Burk双倒数图，计算动力学参数、最大初速度(Vmax)和Michaelis-Menten常数(Km)。
如图4a所示，Lineweaver-Burk双倒数曲线相交于y轴，随着该降压肽质量浓度的增加，Km逐渐升高，而Vmax未发生明显变化，说明它属于一种竞争性抑制肽。由图4c可知，经分子动力学模拟分析发现灵芝菌丝体降压肽Ser-Tyr-Pro通过Ser1氨基与ACE活性区域的Lys472羧基形成一个盐键；通过Ser1羟基与ACE活性区域的Lys472、Tyr2羟基与Gln242、Pro3羧基与Lys415形成氢键；通过Tyr2苯环、Pro3闭环与ACE活性区域的Tyr484、Phe488产生疏水作用，从而占据ACE的活性区域，使其不能作用底物而失活。
实施例3：灵芝菌丝体降压肽的胃肠道消化稳定性分析
向灵芝菌丝体降压肽Ser-Tyr-Pro溶液(pH为2，1mg/mL)中加入1.0％(w/w)胃蛋白酶于37℃反应30min。反应结束后，用1M NaOH调pH至7.5，即得该降压肽的胃液消化液。向该消化液(pH 7.5)中加入1.0％(w/w)胰酶于37℃反应90min，反应结束后于95℃中放置10min使酶失活。然后置于冰水上5min，即得该样品的胃肠液消化产物。以不加胃蛋白酶或胰酶的小肽溶液作为实验对照组，利用RP-HPLC对该降压肽的胃肠液消化稳定性进行分析，并测定其ACE抑制活性。色谱条件：色谱柱为Znertsil ODS-3(4.6×250mm)；流动性为水(含0.04％三氟乙酸)，乙腈(0.03％三氟乙酸)；洗脱条件为0-5min，0％乙腈；5-40min，0-40％乙腈；温度为25℃；检测波长220nm；流速为0.5mL/min。
如图5所示，灵芝菌丝体降压肽Ser-Tyr-Pro能被胃蛋白酶降解，但不被胰酶降解，说明其在肠道具有很好的稳定性。
实施例4：降压肽Ser-Tyr-Pro的体内体外降血压功效分析
1、灵芝菌丝体降压肽的体内降血压功效评价
选用湖南斯莱克景达实验动物有限公司提供的8周、尾部动脉收缩压超过180mmHg的雄性自发性高血压大鼠SHR。饲养于23℃恒温12h光照及12h黑暗处理，食物与水自由采食，适应性饲养1周。将SHR随机分为3组，每组6只，第1组为阴性对照组，注射同等剂量生理盐水，第2组通过静脉注射方式进行给药，第3组组通过腹腔注射方式进行给药。灵芝菌丝体降压肽Ser-Tyr-Pro按照10mg/kg动物体重的规定剂量一次性给药，测定SHR给药后0-8h的尾部动脉收缩压和舒张压，每只大鼠血压值测定5次取平均值。
如图6所示，腹腔注射或静脉注射该降压肽，均能降低SHR舒张压和收缩压。在给药2h后，收缩压和舒张压均显著下降，由于该降压肽在进入血液中被部分代谢降解，当给药3h后血压均有所回升。其中，由图6a可知，在给药2h时，未给药组收缩压为160.7mmHg，相较于腹腔注射，以静脉注射的给药方式降收缩压效果更好，收缩压降低至122.6mmHg；图6b中未给药组舒张压为134.7mmHg，静脉注射比腹腔注射的降压效果更好，舒张压可降低至96.3mmHg.
2、灵芝菌丝体降压肽的体外降血压活性分析
人脐带静脉内皮细胞(HUVEC)用RPMI 1640培养液进行培养，该培养液中还含有10％胎牛血清、100μg/mL青霉素和100μg/mL链霉素。采用显微镜观察及MTS细胞活力测定方法分析降压肽Ser-Tyr-Pro对血管紧张素I诱导HUVEC的eNOS和内皮素-1表达的调控情况。
1)eNOS和内皮素-1蛋白表达调控情况分析：将HUVEC(1×10 5个细胞/mL)种植于10cm细胞培养皿中，在37℃下含有5％CO 2的二氧化碳培养箱中培养。待细胞成熟后小心移去上层液，加入1mLhanks液,移去上层液，重复3次，加入4mL Trypsin-EDTA进行消化，细胞收缩变圆后加入12mL RPMI 1640培养液终止消化。将其分至10cm培养皿中(2mL/皿)，共5皿，每皿补新鲜培养液至10mL/皿。待细胞长满至90％，加入血管紧张素I(1.0mg/mL)诱导2h，建立高血压细胞模型。分别加入不同浓度(0、25、50、100μg/mL)降压肽Ser-Tyr-Pro，其中以不加血管紧张素I和降压肽的为对照组，以不加降压肽的为诱导组，提取总蛋白质利用Wester-blot技术检测eNOS和内皮素-1蛋白表达水平。
由图7c，d和e可知，相较于诱导组，灵芝菌丝体降压肽Ser-Tyr-Pro可上调eNOS磷酸 化水平，抑制内皮素-1蛋白表达水平。其中，当该降压肽的处理剂量为25μg/mL时，磷酸化eNOS蛋白表达水平增加了4.86倍，内皮素-1蛋白表达水平下降了63.9％。
2)eNOS和内皮素-1mRNA表达调控情况分析：将HUVEC(1×10 5个细胞/mL)种植于10cm细胞培养皿中，在37℃下含有5％CO 2的二氧化碳培养箱中培养。待细胞成熟后小心移去上层液，加入1mL hanks液，移去上层液，重复3次，加入4mL Trypsin-EDTA进行消化，细胞收缩变圆后加入12mL RPMI 1640培养液终止消化。将其分至6孔板中(2mL/孔)，共5孔。待细胞长满至80％，加入血管紧张素I(1.0mg/mL)诱导2h，建立高血压细胞模型。分别加入不同浓度(0、25、50、100μg/mL)降压肽Ser-Tyr-Pro，其中以不加血管紧张素I和降压肽的为对照组，以不加降压肽的为诱导组，提取总RNA反转录为cDNA，利用琼脂糖电泳检测其完整性；并利用RT-qPCR技术检测eNOS和内皮素-1mRNA表达水平。
PCR引物设计：
内皮素-1反链：5’CAG AAA CTC CAC CCC TGT GT3’(序列如SEQ ID NO:3所示),
内皮素-1正链：5’TCC TCT GCT GGT TCC TGA CT 3’(序列如SEQ ID NO:4所示)
eNOS反链：5’GGT GGC CCT CGT GGA CTT GC 3’(序列如SEQ ID NO:5所示)
eNOS正链：5’AGG CCT TCC GAG GCT GG 3’(序列如SEQ ID NO:6所示)
β-actin反链：5’CAT GTA CGT TGC TAT CCA GGC 3’(序列如SEQ ID NO:7所示)
β-actin正链：5’CTC CTT AAT GTC ACG CAC GAT 3’(序列如SEQ ID NO:8所示)。
如图7a和b所示，灵芝菌丝体降压肽Ser-Tyr-Pro对eNOS和内皮素-1的mRNA水平调控与其蛋白表达水平的变化趋势相似。相较于诱导组，当该降压肽治疗剂量为25μg/mL时，eNOS的mRNA水平增加了3.51倍，内皮素-1的mRNA水平下降了27.3％。

Claims (10)

  1. 一种肽，其特征在于，所述肽的氨基酸序列为Ser-Tyr-Pro。
  2. 一株灵芝菌株SCIM 1006，所述菌株于2019年11月25日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.18819。
  3. 一种生产权利要求1所述肽的方法，其特征在于，所述方法是采用人工合成法或者生物发酵法或者酶解法进行生产；可选地，所述方法包括利用权利要求2所述的灵芝菌株SCIM1006进行发酵生产。
  4. 根据权利要求3所述的方法，其特征在于，所述发酵使用的培养基为含有混合氨基酸的发酵培养基；可选地，所述发酵培养基中的混合氨基酸包括如下两种以上的氨基酸：Ler、Trp、Val、Tyr、Asp、Phe、Glu、Asn、Thr、Gln、Cys、His、Pro、Ser、Gly、Arg、Ala；可选地，所述发酵，是在温度20-28℃、100-140r/min的条件下，发酵6～8d。
  5. 从灵芝发酵液中提取纯化权利要求1所述肽的方法，其特征在于，所述灵芝发酵液为灵芝菌株SCIM 1006的发酵液；可选地，所述方法，包括：用匀浆缓冲液和超滤膜对灵芝菌丝体中降压肽进行提取分离；通过高效液相色谱纯化，获得灵芝菌丝体降压肽纯品。
  6. 一种具有降血压作用的药物，所述药物中含有权利要求1的氨基酸序列为Ser-Tyr-Pro的肽或者权利要求2所述的灵芝菌株SCIM 1006；可选地，所述药物按常规制剂工艺制备成临床常用的胶囊剂、片剂、颗粒剂、丸剂、散剂或口服液。
  7. 一种非疾病的诊断和治疗的调控eNOS和内皮素-1表达的方法，其特征在于，所述方法包括利用权利要求1所述的肽或者权利要求2所述的灵芝菌株SCIM 1006。
  8. 编码权利要求1所述肽的核苷酸序列。
  9. 表达权利要求1所述肽的载体或重组菌。
  10. 一种试剂盒，其特征在于，所述试剂盒中包括权利要求1所述的肽或者权利要求2所述的灵芝菌株SCIM 1006。
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