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Actinomycete fibrinolytic enzyme and preparation method thereof

An actinomycetes and plasmin technology, applied in biochemical equipment and methods, microorganism-based methods, enzymes, etc., can solve the problems of reducing plasmin activity, decreasing fibrinolytic effect, and many separation and purification steps, etc. The effect of good fibrin dissolving performance, short enzyme production cycle and high enzyme production activity

Inactive Publication Date: 2013-10-02
QIQIHAR UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For nearly ten years, the applicant has been exploring the application potential of microorganisms in the preparation of modern biothrombolytic agents, successfully Two kinds of microbial fibrinolytic enzymes with patent application numbers 200610163497.6 and 200810137564.4 have been cultivated. The strains used for the cultivation of these two kinds of fibrinolytic enzymes are all fungi, and there are many fungal fermentation metabolites, which lead to subsequent separation and purification of fibrinolytic enzymes There are many steps, and too many separation and purification steps will reduce the activity of fibrinolytic enzymes, resulting in a decline in the fibrinolytic effect. Therefore, in recent years, the applicant has been working hard to find excellent enzymes with fewer types of fermentation metabolites and easy product separation and purification. Bacterial classification, the present invention is exactly the part content of this research progress

Method used

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  • Actinomycete fibrinolytic enzyme and preparation method thereof
  • Actinomycete fibrinolytic enzyme and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Strain: Actinomyces strain YY21, the preservation number is CGMCC N O .5816

[0026] 2. Medium and culture conditions:

[0027] (1) Incline medium: glucose 0.4-0.5%, yeast extract 0.3-0.6%, malt extract 0.5-1.5%, calcium carbonate 0.1-0.2%, agar 1.5%, pH6-7.

[0028] Slant culture conditions: culture at 28°C for 4-7 days.

[0029] (2) Seed medium: glucose 0.4-0.5%, yeast extract 0.3-0.6%, malt extract 0.5-1.5%, calcium carbonate 0.1-0.2%, adjust the pH to 6-7, and sterilize for later use.

[0030] Cultivation conditions: the rotation speed is 170-190r / min, 28°C shaking culture for 28-32h, as a liquid seed.

[0031] (3) Fermentation medium: millet flour 6-8%, glucose 2-4%, calcium carbonate 0.1-0.3%, sodium chloride 0.4-0.6%, peptone 0.6-0.7%, pH7.0-7.4, 5% inoculation quantity.

[0032] Culture conditions: 150-170r / min, 90-100h fermentation at 22°C.

Embodiment 2

[0034] 1. Incline cultivation is the same as in Example 1

[0035] 2, seed culture is the same as embodiment 1

[0036] 3, fermentation culture is the same as embodiment 1

[0037] 4. Separation and purification of actinomycete plasmin:

[0038] The liquid fermentation culture was centrifuged at 10000rpm / min, 4°C for 20 min, and the centrifuged supernatant was subjected to fractional salting out with 30%-60% W / V saturation of ammonium sulfate to obtain crude enzyme liquid.

[0039] a. Octyl-Sepharose FF hydrophobic interaction chromatography, the salt saturation of the sample is adjusted to 30%, and after loading the sample is eluted with a gradient of 30-0% W / V ammonium sulfate solution to collect fibrinolytic active components; and

[0040] b. Phenyl-Sepharose HP hydrophobic interaction chromatography, the salt saturation of the sample was adjusted to 15%, and after loading the sample was eluted with a gradient of 15-0% W / V ammonium sulfate solution, and fibr...

Embodiment 3

[0042] 1. Incline cultivation is the same as in Example 1

[0043] 2, seed culture is the same as embodiment 1

[0044] 3, fermentation culture is the same as embodiment 1

[0045] 4. The separation and purification method of actinomycetes plasmin is the same as in Example 2

[0046] 5. Determine the amino acid sequence of the isolated and purified plasmin : 经过Q-TOF2串联质谱测得其中五个肽段的氨基酸序列分别为:A-Q-S-V-P-Y-G-L-S-Q-L-K,其分子量为1289.6644;V-A-V-L-D-S-G-L-D-S-S-H-P-D-L-K,其分子量为1651.8243;N-S-L-E-S-T-A-T-N-L-G-N-P-A-G-A-T-Y-G-K,其分子量为1964.8844;h-p-n-w-T-n-t-n-v-r,其分子量为1237.5844;Y-P-S-V-L-A-V-G-A-V-D-N-G-V-S-N-K,其 The molecular weight is 1688.8043.

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Abstract

The invention discloses actinomycete fibrinolytic enzyme and a preparation method thereof. According to the actinomycete fibrinolytic enzyme, a actinomycete strain YY21 is adopted as a strain, and culture fermentation, 30-60% W / V saturation degree ammonium sulfate salting-out, Octyl-SepharoseFF hydrophobic interaction chromatography, and Phenyl-Sepharose HP hydrophobic interaction chromatography are performed to prepare the actinomycete fibrinolytic enzyme, wherein a molecular weight of the actinomycete fibrinolytic enzyme is about 32000 daltons, and the actinomycete fibrinolytic enzyme has a fibrin dissolving function. According to the present invention, the used strain has characteristics of short enzyme production period and excellent performances, and can produce other substances having bacterial inhibition activity while producing the enzyme; the actinomycete fibrinolytic enzyme is an extracellular enzyme, such that subsequent separation and purification during preparation are easily performed; and only two step hydrophobic interaction chromatography with advantages of rapid flow rate, high capacity and good sample activity storage are required to prepare the high purity product.

Description

technical field [0001] The invention relates to an actinomycete plasmin, and also relates to a method for preparing the plasmin. Background technique [0002] Thromboembolic diseases seriously endanger human life and health. Thrombolytic therapy is currently the main method for the treatment and prevention of thrombotic diseases. It is a modern medical goal to develop new thrombolytic drugs that are efficient, fast, prevent re-embolism, and reduce adverse reactions such as bleeding. urgent need. [0003] Plasmin has the function of dissolving fibrin, which is the main component of thrombus. The production of plasmin by microbial fermentation has the advantages of short production cycle, small footprint and high product content. Therefore, the development of highly efficient and specific microbial plasmin is the main way to produce new thrombolytic drugs. [0004] In the past ten years, the applicant has been exploring the application potential of microorganisms in the prep...

Claims

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Application Information

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IPC IPC(8): C12N9/68C12R1/01
Inventor 邓永平刘晓兰郑喜群艾瑞波
Owner QIQIHAR UNIVERSITY
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