Method for purifying alliinase by double-water-phase separation

An alliinase, separation and purification technology, used in biochemical equipment and methods, enzymes, lyases and other directions, can solve the problems of reducing enzyme activity, enzyme recovery rate, protein denaturation, harsh conditions, etc., and achieves economical separation process. Simple instrument and time saving effect

Inactive Publication Date: 2014-10-01
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, a series of protein purification techniques are mainly used for the purification of alliinase, such as salting out, affinity chromatography, gel filtration, etc. Although these methods can achieve a certain purification of alliinase, there are many shortcomings, such as operation Complicated and harsh conditions, the separation process is easy to denature the protein, reduce the enzyme activity and the recovery rate of the enzyme

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Prepare a two-phase aqueous system with a mass fraction of 7.5% (w / w) PEG3000 / 10% (w / w) ammonium sulfate, pH=5.0, add 5% (v / v) of alliinase crude extract, shake 15min to completely disperse the phases, centrifuge to completely separate the phases, and record the volume of the upper and lower phases. The lower phase was subjected to ultrafiltration to remove salt, and then vacuum freeze-dried to obtain an enzyme purification factor of 5.06 and an enzyme activity recovery rate of 92.2%.

Embodiment 2

[0022] Prepare a two-phase aqueous system with a mass fraction of 17.5% (w / w) PEG3000 / 18% (w / w) ammonium sulfate, pH=5.0, add 5% (v / v) of alliinase crude extract, shake 15min to completely disperse the phases, centrifuge to completely separate the phases, and record the volume of the upper and lower phases. The lower phase was subjected to ultrafiltration to remove salt, and then vacuum freeze-dried to obtain an enzyme purification factor of 5.58 and an enzyme activity recovery rate of 94.4%.

Embodiment 3

[0024] Prepare a two-phase aqueous system with a mass fraction of 15% (w / w) PEG3000 / 14% (w / w) ammonium sulfate, pH=5.0, add 5% (v / v) of alliinase crude extract, shake 15min to completely disperse the phases, centrifuge to completely separate the phases, and record the volume of the upper and lower phases. The lower phase was subjected to ultrafiltration to remove salt, and then vacuum freeze-dried to obtain an enzyme purification factor of 5.95 and an enzyme activity recovery rate of 96.2%.

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PUM

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Abstract

The invention belongs to the field of deep processing of garlic, and particularly discloses a method for purifying alliinase by double-water-phase separation. The method comprises the following steps: (1) peeling and precooling garlic squamous bulbs, immersing in a precooled extract, homogenizing, centrifuging, and discarding garlic slag, thereby obtaining the supernate crude enzyme solution; (2) dissolving ammonium sulfate, sodium dihydrogen phosphate or trisodium citrate in a Britton-Robinson buffer solution, oscillating to completely dissolve, adding a PEG (polyethylene glycol) solution, evenly mixing by oscillation, and standing for phase splitting, thereby obtaining a double-water-phase extractant; (3) adding the crude enzyme solution into the double-water-phase extractant, oscillating, centrifuging, and standing for phase splitting; and (4) ultrafiltering the lower phase of the enriched alliinase, and carrying out vacuum freeze drying on the trapped fluid to obtain the alliinase solid. The method is simple to operate, has the advantages of simple apparatuses, time saving, low cost, and higher alliinase purification multiple and enzyme activity recovery rate than the prior art, and can easily implement scale-up production.

Description

(1) Technical field [0001] The invention belongs to the field of deep processing of garlic, in particular to a method for separating and purifying alliinase in two aqueous phases. (2) Background technology [0002] Alliinase is an endogenous enzyme present in garlic. Its full name is S-alkyl-L-cysteine ​​sulfoxide, which accounts for about 10-12% of the soluble protein in garlic. It consists of two identical Each subunit is composed of 448 amino acids. Alliinase and its natural substrate alliin are located in different parts of garlic. Alliinase is located in the vacuolar tissue, and the substrate is located in the cytoplasm. When When garlic is broken, the thiosulfinate produced by the contact of alliin and alliinase has anti-cancer, anti-oxidation, anti-atherosclerosis effects, etc., and can be used to prepare thiosulfinate with alliinase and alliinase Sulfinate is used in the production of garlic capsules, so the separation and purification of alliinase is very impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88
CPCC12N9/88C12Y404/01004
Inventor 崔波姜秀敏檀琮萍卢艳敏
Owner QILU UNIV OF TECH
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