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Purification method of MTGase (Microbial Transglutaminase) crude enzyme

A purification method and crude enzyme technology, applied in the field of MTGase crude enzyme purification, can solve the problems of low sample recovery rate, high cost, complicated process, etc., and achieve the effects of high enzyme activity recovery rate, high enzyme activity, and high enzyme purity.

Active Publication Date: 2017-08-18
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the purification method of MTGase generally adopts the method of ion exchange chromatography or gel column chromatography. When those skilled in the art use the two-phase extraction technology to purify the enzyme, they generally choose PEG with a smaller molecular weight such as PEG4000, PEG2000, PEG1000, etc., and extract At the same time, the enzyme to be purified will be distributed in the PEG-rich upper phase. In order to separate the PEG from the enzyme to be purified, back extraction is required, and the enzyme to be purified will be extracted into the salt phase (lower phase), so that the production process Complexity, high cost, cumbersome process, low sample recovery rate, expensive separation materials and equipment, small sample processing volume, low separation purity of a single method, etc., it is difficult to meet the needs of production and application

Method used

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  • Purification method of MTGase (Microbial Transglutaminase) crude enzyme

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Embodiment 1

[0030] The purification method of MTGase crude enzyme, comprises the following steps:

[0031] The crude enzyme powder of MTGase is dissolved in water, is mixed with 2wt% MTGase crude enzyme liquid;

[0032] Prepare PEG-6000 / (NH 4 ) 2 SO 4 A two-phase aqueous solution consisting of:

[0033] PEG-6000 27wt%

[0034] (NH 4 ) 2 SO 4 13wt%

[0035] Water 60wt%;

[0036] PEG-6000 / (NH 4 ) 2 SO 4 The two-phase aqueous solution forms the PEG-6000 phase of the upper layer and the salt phase of the lower layer;

[0037] Mix MTGase crude enzyme solution with PEG-6000 / (NH 4 ) 2 SO 4 The aqueous two-phase solution was mixed uniformly according to the mass ratio of 1:5, and stood at 30°C for 1 h, MTGase entered the salt phase in the two-phase system, and collected the salt phase in the two-phase system;

[0038] The collected salt phase is pumped into the nanofiltration membrane equipment for processing. The nanofiltration membrane material used is polyacrylonitrile, the mo...

Embodiment 2

[0040]The purification method of MTGase crude enzyme, comprises the following steps:

[0041] The crude enzyme powder of MTGase is dissolved in water, is mixed with 2wt% MTGase crude enzyme liquid;

[0042] Prepare PEG-8000 / (NH 4 ) 2 SO 4 A two-phase aqueous solution consisting of:

[0043] PEG-10000 30wt%

[0044] (NH 4 ) 2 SO 4 12wt%

[0045] Water 58wt%;

[0046] PEG-8000 / (NH 4 ) 2 SO 4 The two-phase aqueous solution forms the PEG-8000 phase of the upper layer and the salt phase of the lower layer;

[0047] Mix MTGase crude enzyme solution with PEG-8000 / (NH 4 ) 2 SO 4 The aqueous two-phase solution was mixed uniformly according to the mass ratio of 1:5, and stood at 20°C for 1 h, MTGase entered the salt phase in the two-phase system, and collected the salt phase in the two-phase system;

[0048] The collected salt phase is pumped into the nanofiltration membrane equipment for processing. The nanofiltration membrane material used is polyacrylonitrile, the mo...

Embodiment 3

[0050] The purification method of MTGase crude enzyme, comprises the following steps:

[0051] The crude enzyme powder of MTGase is dissolved in water, is mixed with 1.8wt% MTGase crude enzyme liquid;

[0052] Prepare PEG-10000 / (NH 4 ) 2 SO 4 A two-phase aqueous solution consisting of:

[0053] PEG-10000 25wt%

[0054] (NH 4 ) 2 SO 4 20wt%

[0055] Water 55wt%;

[0056] PEG-10000 / (NH 4 ) 2 SO 4 The two-phase aqueous solution forms the PEG-10000 phase of the upper layer and the salt phase of the lower layer;

[0057] Mix MTGase crude enzyme solution with PEG / (NH 4 ) 2 SO 4 The aqueous two-phase solution was mixed uniformly according to the mass ratio of 1:3, and stood at 40°C for 1 hour, MTGase entered the salt phase in the two-phase system, and collected the salt phase in the two-phase system;

[0058] The collected salt phase is pumped into the nanofiltration membrane equipment for processing. The nanofiltration membrane material used is polyacrylonitrile, th...

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Abstract

The invention discloses a purification method of an MTGase (Microbial Transglutaminase) crude enzyme. The purification method comprises the following steps: dissolving MTGase crude enzyme powder with water to prepare an MTGase crude enzyme solution with the concentration of 1 weight percent to 2 weight percent; preparing a PEG (Polyethylene Glycol) / (NH4)2SO4 double-water-phase solution to form an upper-layer PEG phase and a lower-layer salt phase; uniformly mixing the MTGase crude enzyme solution with the PEG / (NH4)2SO4 double-water-phase solution; standing at 20 DEG C to 40 DEG C for 1h to 2h; enabling MTGase to enter the salt phase in a double-water-phase system and collecting the salt phase in the double-water-phase system; carrying out nanofiltration membrane treatment on the collected salt phase and collecting inter-membrane liquid of the nanofiltration membrane treatment, so as to obtain purified MTGase. The purification method has the advantages that purification is simple and convenient, the purity of the MTGase obtained by purification and separation is relatively high and the recycling rate is high.

Description

technical field [0001] The invention relates to an enzyme purification method, in particular to a purification method of MTGase crude enzyme. Background technique [0002] Transglutaminase (TGase for short) is a transferase that catalyzes amidotransfer reaction. The hydrolysis reaction can directly change the structure and functional properties of the protein itself and the cells and tissues to which the protein is attached, improve the nutritional value of the protein, and produce a new type of protein food that meets people's needs. The enzyme activity of TGase (MTGase) derived from microorganisms is completely independent of the presence of calcium ions. It has a small molecular weight and is easier to store at room temperature. It is similar in function to TGase derived from animals. These properties make microbial transglutaminase useful in industrial production and The application prospects are huge. At present, the "National Food Safety Standard Food Additive Use St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10
CPCC12N9/1044C12Y203/02013
Inventor 章骞曹敏杰刘敏莉翁凌张凌晶刘光明
Owner JIMEI UNIV
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