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Separation and purification method of nattokinase

A technique for separation and purification of nattokinase, applied in peptide preparation methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as increased ionic strength, interference in purification steps, low recovery rate, etc., and achieve relief The effect of high salt ion concentration, simplified purification steps, and reduced enzyme loss

Pending Publication Date: 2022-03-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since ammonium sulfate precipitates, the ionic strength of the entire enzyme solution increases. When using an ion exchange column, dialysis and desalination are generally required first, otherwise it will cause great interference to the subsequent purification steps, so the entire process often takes a long time.
[0008] Shi Yanmao and others used CM-52 cation exchange chromatography for separation, and the recovery rate of the enzyme activity was only 48.51%; Wang Gang and others used ammonium sulfate to precipitate the nattokinase fermentation broth, and the salted-out product was filtered with SephadexG-50 gel , its recovery rate is 42.1%, and SephadexG-50 has only molecular sieve effect but no ion exchange capacity, and the effect is relatively poor; there are some defects in this multi-step treatment method, such as complicated steps, long time consumption, low recovery rate, etc. However, the anion exchange chromatography used in it can greatly improve the specific activity of the product.

Method used

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  • Separation and purification method of nattokinase
  • Separation and purification method of nattokinase
  • Separation and purification method of nattokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Preparation of Nattokinase Crude Product and Detection of Protein Content and Enzyme Activity

[0044] 1. Preparation of nattokinase crude enzyme

[0045] Nattokinase is obtained by fermentation of Bacillus cereus (Bacillus cereus) CGMCC No 12336. The specific preparation process is as follows: soybean selection → cleaning and soaking → sterilization and steaming → inoculation → fermentation → post-ripening at 4°C → nattokinase bacterial liquid, process The main points are as follows:

[0046] (1) Natto matrix: Soybeans are commercially available soybeans with full granules. Wash the purchased soybeans with water, soak them at room temperature for 6 hours, control the moisture, and put 100g / bottle into five 250mL triangular flasks. Then add 3g NaCl and 3g sucrose to each bottle, seal it with eight layers of gauze and steam it (sterilized at 121°C for 20 minutes).

[0047] (2) Fermentation of natto: Pick a ring of colonies from the inclined plane of Bacillus...

Embodiment 2

[0067] Embodiment 2: Purification of nattokinase by ion exchange chromatography

[0068] The isoelectric point of nattokinase is 8.6, stable in property when pH6-12, selects anion-exchange resin DEAE-Sephadex A-50 (recorded as A-50) as chromatographic medium, 0.1M pH7.4 phosphate buffer for washing Deliquification; Anion-exchange resin DEAE-Sephadex CL-6B (GE Reagent Company) was selected as a contrast chromatographic medium in addition, and the conditions such as its packing height, balance and sample amount elution were all the same as those of the DEAE-Sephadex A-50 layer The analysis medium is the same.

[0069] 1. Anion exchange chromatography medium pretreatment:

[0070] Stir 5g of A-50 evenly and soak in 500mL of pure water. After 1h, pour out the floating particles in the upper layer. Then soak A-50 in 0.5M NaOH aqueous solution according to the ratio of adding 15mL of 0.5M NaOH aqueous solution per gram of A-50, shake well, let it stand at room temperature for 0.5h...

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Abstract

The invention discloses a separation and purification method of nattokinase, which comprises the following steps: dissolving a nattokinase crude product with a buffer solution, filtering, and carrying out DEAE-Sephadex A-50 anion exchange column chromatography on the filtrate as a loading solution; after the sample loading is finished, eluting with a 0.1 M phosphate buffer solution with the pH value of 7.4 at the elution speed of 10s / drop, monitoring the effluent by adopting a chromatographic workstation, collecting according to every 10min / tube before peak appearance, collecting at each peak at the peak appearance position, and eluting until the effluent does not appear; and combining the effluent containing nattokinase protein and enzyme activity, carrying out ultrafiltration, and freeze-drying the filtrate to obtain the nattokinase pure enzyme. According to the method disclosed by the invention, the purification steps are simplified, the enzyme loss in the purification process is reduced, the time and cost required by purification are greatly reduced, the specific activity of the electrophoresis pure nattokinase reaches 90080.6 U / mg, the purification multiple is 3.2, and the enzyme activity recovery rate is 82.62%.

Description

(1) Technical field [0001] The invention relates to a method for purifying nattokinase, in particular to a method for separating and purifying nattokinase from mixed samples by using DEAE-Sephadex A-50 anion exchange chromatography resin. (2) Background technology [0002] Thrombotic disease is a class of diseases that can seriously threaten human health and life, including common diseases such as heart and cerebral thrombosis and pulmonary embolism. It is estimated that more than 10 million people in the world suffer from thrombotic diseases; in my country, with the improvement of material living conditions and the aging of the population, the number of patients with thrombotic diseases is increasing year by year, and a large number of people die every year. Therefore major countries in the world are devoting themselves to the research and development of drugs for the treatment of thrombotic diseases. [0003] Serine proteases are a class of proteases named after serine re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/56C07K1/18C07K1/34C07K1/14
CPCC12N9/54C12Y304/21062
Inventor 欧志敏程朋朋卢媛王金美张楚玥
Owner ZHEJIANG UNIV OF TECH