Fibrinolysin of fermented soya beans and culture method thereof

Abstract

The invention relates to fibrinolysin of fermented soya beans and a culture method thereof. The fibrinolysin of fermented soya beans is prepared through fermentation, separation and purification on a strain Jxnuwx-1 as a culture strain, wherein the preservation number of the strain in the fermented soya beans is CCTCC M2014638 and the taxonomic name of the strain is Bacillus amyloliquefaciens. The fibrinolysin of fermented soya beans is fibrinolysin, the relative molecular mass of which is 29kDa. The optimum action temperature is 41 DEG C, the temperature stabilizing range is 23-43 DEG C, the optimum pH is 7.6, and the pH stabilizing interval is 7.0-11.0. The fibrinolysin of fermented soya bean can successively degrade Aalpha, Bbeta and Cgamma chains of human fibrin (proteinogen) within 4h. The fibrinolysin of fermented soya beans not only can degrade fibrous protein and fibrinogen, but also can serve as a plasminogen activating factor to degrade thrombin. The influence of the fibrinolysin of fermented soya beans on proteins in a human blood is relatively small, and the fibrinolysin of fermented soya beans provides a novel path for development and utilization of antithrombotic drugs.

Description

technical field

[0001] The present invention relates to fermented soya bean fibrinolytic enzyme, and also relates to a method for cultivating fermented soya bean fibrinolytic enzyme. Background technique

[0002] Cardiovascular disease is a common and fatal disease, accounting for about 29% of global mortality, and the formation of thrombus is one of the main causes of cardiovascular disease, and the formation of thrombus is a very complicated physiological process. The main component of thrombus is fibrin, and the excessive accumulation of fibrin in blood vessels will easily form thrombus. Plasmin can degrade fibrin. Common plasmin agents include urokinase type (U-PA), tissue type (T-PA), bacterial type and streptokinase type. Although plasmin has a significant effect on the treatment of thrombus, However, it also has certain defects, such as the market is in short supply, the price is too expensive, the specificity of degrading fibrin is poor, causing internal bleeding, a...

Examples

Embodiment 1

[0023] Example 1: Preparation of liquid fermentation culture

[0024] Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain Jxnuwx-1 (preservation number: CCTCC M 2014638) was used as the culture strain to carry out slant culture, seed culture and liquid fermentation culture, and the liquid fermentation culture was obtained after 3 days of fermentation.

[0025] Wherein, culture medium and culture condition:

[0026] (1) Incline medium: beef extract 0.3%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.0. The sterilization temperature of the culture medium is 121°C, and the sterilization time is 30min. The culture condition was 37°C, the culture time was 24h, and then the slant was preserved at 4°C.

[0027] (2) Seed medium: 5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0. The sterilization temperature of the medium is 115°C, and the sterilization time is 30min. The culture conditions were shake flask culture, 170r / min, 12h, 37°C.

[0028] (3) Fermentation medium: 2% sucrose...

Embodiment 2

[0029] Embodiment 2: Separation and purification of tempeh fibrinolytic enzyme

[0030] The liquid fermentation culture obtained in Example 1 was subjected to 65% saturation ammonium sulfate salting out and precipitation, and was suspended with a phosphate buffer solution with a pH of 7.0 to obtain a crude enzyme solution, and then passed through:

[0031] a. Use a dialysis bag to perform dialysis and desalination while performing buffer exchange. The dialysis buffer is Tris-HCl buffer with a pH of 8.5 and a concentration of 20 mM.

[0032] b. Put the desalted solution containing plasmin on the DEAE-sepHarose FF column, use 0-0.5moL / L NaCl and Tris-HCl buffer solution with a pH of 8.5, and the concentration is 20mM to elute, and collect the fibrinolytic active group point.

[0033] c. Then perform gel chromatography with Superdex 75 column, elute with phosphate buffer solution with pH 7.0, the eluent contains 0.3moL / L NaCl, collect fibrinolytic active ingredients, and obtain ...

Embodiment 3

[0034] Embodiment 3: Determining the properties of the fermented soya bean fibrinolytic enzyme after separation and purification

[0035] Using the SDS-PAGE method to determine the relative molecular mass of the fermented soybean fibrinolytic enzyme obtained in Example 2 is 29kDa, and the optimum temperature and optimum pH are respectively 41°C and 7.6. , Fe 3+ , Fe 2+ Inhibited, the tempeh plasmin can rapidly degrade the Aα subunit of fibrin (ogen), followed by the Bβ and Cγ subunits, the tempeh plasmin can not only directly degrade fibrin (ogen) but also act as a The original activator, while the tempeh plasmin can also degrade thrombin.

technical field

[0001] The present invention relates to fermented soya bean fibrinolytic enzyme, and also relates to a method for cultivating fermented soya bean fibrinolytic enzyme. Background technique

[0002] Cardiovascular disease is a common and fatal disease, accounting for about 29% of global mortality, and the formation of thrombus is one of the main causes of cardiovascular disease, and the formation of thrombus is a very complicated physiological process. The main component of thrombus is fibrin, and the excessive accumulation of fibrin in blood vessels will easily form thrombus. Plasmin can degrade fibrin. Common plasmin agents include urokinase type (U-PA), tissue type (T-PA), bacterial type and streptokinase type. Although plasmin has a significant effect on the treatment of thrombus, However, it also has certain defects, such as the market is in short supply, the price is too expensive, the specificity of degrading fibrin is poor, causing internal bleeding, a...

Examples

Embodiment 1

[0023] Example 1: Preparation of liquid fermentation culture

[0024] Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain Jxnuwx-1 (preservation number: CCTCC M 2014638) was used as the culture strain to carry out slant culture, seed culture and liquid fermentation culture, and the liquid fermentation culture was obtained after 3 days of fermentation.

[0025] Wherein, culture medium and culture condition:

[0026] (1) Incline medium: beef extract 0.3%, peptone 1%, NaCl 0.5%, agar 2%, pH 7.0. The sterilization temperature of the culture medium is 121°C, and the sterilization time is 30min. The culture condition was 37°C, the culture time was 24h, and then the slant was preserved at 4°C.

[0027] (2) Seed medium: 5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0. The sterilization temperature of the medium is 115°C, and the sterilization time is 30min. The culture conditions were shake flask culture, 170r / min, 12h, 37°C.

[0028] (3) Fermentation medium: 2% sucrose...

Embodiment 2

[0029] Embodiment 2: Separation and purification of tempeh fibrinolytic enzyme

[0030] The liquid fermentation culture obtained in Example 1 was subjected to 65% saturation ammonium sulfate salting out and precipitation, and was suspended with a phosphate buffer solution with a pH of 7.0 to obtain a crude enzyme solution, and then passed through:

[0031] a. Use a dialysis bag to perform dialysis and desalination while performing buffer exchange. The dialysis buffer is Tris-HCl buffer with a pH of 8.5 and a concentration of 20 mM.

[0032] b. Put the desalted solution containing plasmin on the DEAE-sepHarose FF column, use 0-0.5moL / L NaCl and Tris-HCl buffer solution with a pH of 8.5, and the concentration is 20mM to elute, and collect the fibrinolytic active group point.

[0033] c. Then perform gel chromatography with Superdex 75 column, elute with phosphate buffer solution with pH 7.0, the eluent contains 0.3moL / L NaCl, collect fibrinolytic active ingredients, and obtain ...

Embodiment 3

[0034] Embodiment 3: Determining the properties of the fermented soya bean fibrinolytic enzyme after separation and purification

[0035] Using the SDS-PAGE method to determine the relative molecular mass of the fermented soybean fibrinolytic enzyme obtained in Example 2 is 29kDa, and the optimum temperature and optimum pH are respectively 41°C and 7.6. , Fe 3+ , Fe 2+ Inhibited, the tempeh plasmin can rapidly degrade the Aα subunit of fibrin (ogen), followed by the Bβ and Cγ subunits, the tempeh plasmin can not only directly degrade fibrin (ogen) but also act as a The original activator, while the tempeh plasmin can also degrade thrombin.

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