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Method for separating and purifying recombinant bacillus subtilis fibrinolytic enzyme

A Bacillus subtilis, separation and purification technology, applied in the field of protein separation and purification, can solve the problems of Bacillus subtilis plasmin separation and purification, high purification cost, low solid fermentation content, etc., achieves huge market application value, and is easy to industrialize amplification , the effect of huge development potential

Pending Publication Date: 2016-11-23
WUHAN ZHENFU PHARMA CO LTD
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Problems solved by technology

However, the key technology for the separation and purification of this protein has not been effectively resolved, and the separation and purification of a large amount of highly purified Bacillus subtilis plasmin is a necessary prerequisite for the development and industrialization of Bacillus subtilis plasmin drugs
Since the discovery of Bacillus subtilis fibrinolytic enzyme in the 1980s, it has been difficult to achieve a large amount of separation and purification of Bacillus subtilis fibrinolytic enzyme by relying on traditional soybean solid fermentation.
Due to the extremely low solid fermentation content and the extremely complex fermentation products, the purification cost is bound to be extremely high
Therefore, Bacillus subtilis fibrinolytic enzyme has been used in the development of health food field, and has not exerted its own important medicinal value

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  • Method for separating and purifying recombinant bacillus subtilis fibrinolytic enzyme

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Embodiment 1

[0028] As shown in Table 1, according to the characteristics of the recombinant Bacillus subtilis plasmin protein, according to the purification efficiency and final protein purification purity, the types of phenyl sepharose series hydrophobic fillers can be selected from the following types: Phenyl Sepharose 6FF (HS), Octyl Sepharose FF, CM-Sepharose FF, Benzamidine 4FF (HS), Phenyl Sepharose 6FF (HS) and Benzamidine 4FF (HS) have a purity of more than 95%. Considering the cost factor, the final comprehensive comparison selection determines that the purified filler is Phenyl Sepharose 6FF(HS). This embodiment is illustrated by taking Phenyl Sepharose6FF (HS) as an example.

[0029] A method for separating and purifying recombinant Bacillus subtilis plasmin, comprising the following steps:

[0030] 1) Hydrophobic chromatography: choose Phenyl Sepharose 6FF (HS) to fill the chromatographic column, the mobile phase buffer is liquid A and liquid B, adjust the purification equipm...

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Abstract

The invention provides a method for separating and purifying recombinant bacillus subtilis fibrinolytic enzyme. The method comprises the following steps: carrying out hydrophobic chromatography: filling a chromatographic column with phenyl-agarose-series hydrophobic filler, washing and balancing the chromatographic column with liquid A, detecting when an ultraviolet absorption wavelength is 280 nanometers, removing thallus of fermentation liquor of the recombinant bacillus subtilis fibrinolytic enzyme and then directly loading a sample into the hydrophobic chromatographic column, continuously adding the liquid A in a flowing way until a flow-through peak flows through completely, and replacing the liquid A with liquid B for eluting until the recombinant bacillus subtilis fibrinolytic enzyme combined with the filler is completely eluted so as to obtain the high-purity recombinant bacillus subtilis fibrinolytic enzyme. A bacillus subtilis fibrinolytic enzyme separation and purification method with a pilot scale test level is established in a fermentation culture solution on the basis of recombinant yeast fermentation of the bacillus subtilis fibrinolytic enzyme; after the method is used, a great deal of high-purity fibrinolytic enzyme can be obtained, and the purity of the obtained fibrinolytic enzyme reaches up to 95%.

Description

technical field [0001] The invention belongs to the field of separation and purification of proteins, in particular to a method for separation and purification of recombinant Bacillus subtilis plasmin. Background technique [0002] my country has a large population, the aging rate of the population is accelerating, the morbidity and mortality of cardiovascular and cerebrovascular diseases are constantly increasing, and the incidence of cardiovascular and cerebrovascular diseases is becoming more and more serious. The number of deaths due to cardiovascular and cerebrovascular diseases is as high as 700 every year The number of patients with cardiovascular and cerebrovascular diseases has exceeded 300 million, and has become the number one killer of human health. In my country alone, the market related to cardiovascular and cerebrovascular diseases has exceeded 1 trillion yuan. The main cause behind cardiovascular and cerebrovascular diseases is the formation of thrombus. How...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68C12Q1/34
Inventor 王业富董艳山高丽石毅向好
Owner WUHAN ZHENFU PHARMA CO LTD
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