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Industrial production method of earthworm fibrinolytic enzyme directly extracted by utilizing ion exchange resin

A technology of ion exchange resin and production method, applied in the biological field, can solve the problems of low product purity and biological activity, high production cost, low yield of earthworm fibrinolytic enzyme production process, etc., and achieve reduction of equipment volume and chemical raw material consumption , production cost reduction, and the effect of improving specificity

Inactive Publication Date: 2010-12-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims at the disadvantages of the current industrial production process of earthworm fibrinolytic enzyme, such as low yield, low product purity and biological activity, high production cost, and the need to consume a large amount of chemical raw materials such as ammonium sulfate or ethanol, acetone, etc., and discloses a simple and efficient Industrialized preparation technology of earthworm fibrinolytic enzyme

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 22Kg of fresh baby Aisheng worm, prepared 0.14mol / L NaCl solution, earthworm homogenate, added 22L of 0.14mol / L NaCl solution in total, stirred and extracted for 2 hours, centrifuged at 5000g, 10°C for 45 minutes, and took the supernatant. Add 5L of 0.14mol / L NaCl solution to the precipitate, stir and extract for 0.5 hours, centrifuge, and combine the supernatants of the two centrifugations. Adjust the pH to 8.5 with 1mol / L NaOH, add deionized water to adjust the conductivity to 2.3ml / Ω, add 4.4Kg of activated D254 resin (macroporous strong basic anion exchange resin), stir for 4 hours, and drain. Rinse thoroughly with normal water, drain, and repeat the rinsing once. Add 10L of 1mol / L NaCl solution to the resin, stir and wash for 2 hours, drain and repeat the washing for 1 hour. Add 6L of 3.5mol / L NaCl solution (pH 5.5) to the drained resin, stir and elute for 4 hours, and drain; similarly stir and elute for 2 hours, drain and combine 2 eluents, suction filtration (th...

Embodiment 2

[0038] Frozen fresh Aisheng worm 28Kg, homogenized, extracted twice with 0.14mol / L NaCl as above, and shared 34L of NaCl solution. The crude earthworm extract was adjusted to pH 9.0 with 1 mol / L NaOH solution, and deionized water was added to adjust the conductivity to 1.8 ml / Ω. Activated ZGA451sc resin (macroporous weakly basic anion exchange resin) 6kg, packed with cross column - earthworm crude extract loading - deionized water washing - 0.5mol / L, 4.5mol / L NaCl solution (pH5.3) each 8L, gradient elution. Following a large and gentle elution peak of impurity protein, a connected peak of plasmin activity appeared, and the elution peak of plasmin was collected and combined, and filtered by suction. The filtrate was ultra-filtered through a hollow fiber ultra-filter (molecular weight cut-off 10,000 D.), washed with deionized water, and concentrated. The concentrate was freeze-dried to obtain 104.4 g of white, odorless plasmin powder.

[0039] Using the (bovine blood) fibrin ...

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Abstract

The invention relates to an industrial production method of earthworm fibrinolytic enzyme directly extracted by utilizing ion exchange resin, belonging to the field of biotechnology. Fresh earthworms or refrigerated earthworms are added into 0.14mol / L of NaC1 for homogenating, stirring and extracting, supernatant fluid is centrifugated, the pH value is adjusted to be 7.5-9.5 and the electrical conductivity is 1-5ml / ohm; activated macroporous negative ion exchange resin is added for stirring and exchanging for 2-4h, then the mixture is washed and eluted in 3-4mol / L of NaC1; or resin is loaded and separated into a column and is exchanged dynamically and is eluted in 0.5-4.5mol / L NaC1 in a gradient; and the earthworm fibrinolytic enzyme is obtained by eluent cooled alcohol precipitation or super filter desalinization and concentration and drying at low temperature. The product of the invention is white, has no peculiar smell, is easy to dissolve in water, improves the biological activityfrom no smaller than 12uku / mg to over 40uku / mg, improves the yield by over 10 percent, saves a great number of chemical raw materials, and lowers the production cost by about 50 percent. The method is suitable for industrial production.

Description

1. Technical field [0001] The invention relates to an industrialized production method for directly extracting earthworm plasmin by utilizing ion exchange resin, and is an industrialized production technology for directly extracting earthworm plasmin by utilizing ion exchange resin as an active ingredient of raw medicine or health food, belonging to biological technical field. 2. Background technology [0002] Select suitable resins to perform ion exchange or adsorption chromatography under appropriate conditions, and directly extract from crude extracts of animals, plants or microorganisms, and directly from fermentation broth to concentrate target proteins or target polysaccharides or other biochemical substances. It is a fast, simple and low-cost industrial separation and purification technology. [0003] Earthworm plasmin, also known as lumbrokinase, has been used for many years in my country and Japan as the main raw material for oral thrombolytic drugs. At the same ti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/68
Inventor 张治国连桂芳
Owner NANJING AGRICULTURAL UNIVERSITY
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