Method for separating purified protamine using reverse micelle method

A technology for separation and purification of protamine, applied in the field of separation and purification of protamine, can solve the problems of long cycle time and complex protamine purification process, and achieve the effect of less separation steps, low cost and easy scale-up

Inactive Publication Date: 2009-02-04
HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for separating and purifying protamine by using the reverse micelles method, extracting and purifying protamine from the crude extract of protamine prepared from milt, and this method can solve the problem of protamine The protein purification process is complicated and the cycle is long

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Separation and Purification of Carp Protamine from Crude Carp Protamine Extract

[0027] (1) Preparation of crude carp protamine extract: Weigh 100g of thawed carp milt in a tissue masher, add 200ml of 0.14M NaCl solution, homogenize for 1 min, filter with gauze to obtain the slurry, Then centrifuge at 8000r / min for 10min, discard the supernatant, and obtain the precipitate, which is the complex DNP of protamine and DNA. Add 4ml of 7.5% sulfuric acid per gram of DNP, stir and mix well, centrifuge at 8000r / min for 10min, discard the precipitate, and the supernatant is the crude carp protamine extract.

[0028] (2) Prepare the reverse micelle solution, and the proportion by weight is AOT:octane=20:878, weigh AOT and octane respectively, take 4.44g sodium disuccinate sulfonate (namely AOT) and 195.56g After octane, mix it evenly and let it stand until it is completely dissolved to obtain 200 g of reverse micelle solution.

[0029] AOT is a surfactant, namely sodium bis(2-ethyl...

Embodiment 2

[0034] Separation and Purification of Salmon Protamine from Crude Salmon Protamine Extract

[0035] (1) Preparation of crude salmon protamine extract: Weigh 100g of salmon white in a tissue masher, add 200ml 0.14M NaCl solution, homogenize for 1 minute, stir in an ice bath for 20 minutes, Let it stand for 10 min, 4000r / min, centrifuge for 10 min, pour out the supernatant, and repeat the operation once more for precipitation. The precipitate was mashed, 4ml of 7.5% sulfuric acid was added to each gram of precipitate DNP, the centrifuge was centrifuged at 4000r / min for 10min, the supernatant was filtered, and the centrifuged precipitate was repeated once again. Combine the two extracts, vacuum filter, and the filtrate is the crude salmon protamine extract.

[0036] (2) Prepare reverse micelle solution: take 22g AOT and 178g n-hexane, the weight ratio of which is AOT: n-hexane=100:800, weigh AOT and n-hexane according to the above ratio, mix them evenly, Leave it until it is complete...

Embodiment 3

[0041] Separation and Purification of Silver Carp Protamine from Crude Silver Carp Protamine Extract

[0042] (1) Prepare silver carp protamine crude extract: Weigh 100g silver carp milt in a tissue masher, add 200ml 0.2M NaCl solution, homogenize for 1 minute, stir in an ice bath for 10 minutes, stand for 5 minutes, 5000r / min, centrifugal separation for 10min, pour out the supernatant, sediment and repeat the operation again. The precipitate was mashed, and 4ml of 7.5% sulfuric acid was added to each gram of precipitate DNP. The centrifuge was centrifuged at 4000r / min for 10min, 4000r / min, centrifuged for 10min, the supernatant was filtered, and the centrifuged precipitate was repeated once again. The two extracts were combined, vacuum filtered, and the filtrate was the crude silver carp protamine extract.

[0043] (2) Prepare the reverse micelle solution: take 8g AOT, 3g Tween 80 and 189g octane, the weight ratio of which is AOT:Tween 80:octane=40:15:945. Weigh AOT, Tween 80 an...

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Abstract

The invention belongs to the technical field of separation and purification of bio-active substances, more particularly relates to a method for directly separating and purifying the protamine from crude protamine extract with a reverse micelle method. The invention aims at solving the problems of complex process, multiple steps and long cycle of present protamine purification. The invention directly separates, purifies the protamine from the crude protamine extract by adopting the reverse micelle solution which consists of a surfactant and an organic solvent and can realize the high-efficient extraction and back extraction of the protamine by adjusting the conditions of extraction processes such as pH of water phases, ionic strength, phase ratio, contact time of two phases; the highest enzyme recovery rate can reach more than 96 percent and the purification factor reaches more than 2.1. The process has the effects of both concentration and decoloration. In addition, the separation and purification of the protamine with the reverse protamine technology have the advantages of short cycle, convenient continuous operation and being easily amplified.

Description

Technical field [0001] The invention belongs to the technical field of separation and purification of biologically active substances, and particularly relates to a method for directly separating and purifying protamine from a crude protamine extract using reverse micelle extraction technology. Background technique [0002] Protamine is a kind of basic protein extracted from the milt tissue of fish. In milt, protamine is combined with DNA in a ratio of 2:1. Protamine is a small and simple spherical basic protein, generally composed of 30-50 amino acids, with a relatively small molecular weight, generally around 6 000 to 10 000 Daltons. The biggest feature of its amino acid composition is that basic amino acids occupies a large proportion, so its basicity is very strong, and its isoelectric point is 10-12. Protamine is soluble in water and dilute acid, can be precipitated by dilute ammonia water, does not solidify when heated, and has good thermal stability. [0003] Toxicological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 刘俊果
Owner HEBEI UNIVERSITY OF SCIENCE AND TECHNOLOGY
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