CN102134564A - Method for separating phospholipase A2 from animal muscles - Google Patents

Method for separating phospholipase A2 from animal muscles Download PDF

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Publication number
CN102134564A
CN102134564A CN 201110003353 CN201110003353A CN102134564A CN 102134564 A CN102134564 A CN 102134564A CN 201110003353 CN201110003353 CN 201110003353 CN 201110003353 A CN201110003353 A CN 201110003353A CN 102134564 A CN102134564 A CN 102134564A
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CN
China
Prior art keywords
phospholipase
tris
damping fluid
hcl damping
animal
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CN 201110003353
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Chinese (zh)
Inventor
王道营
张露娟
卞欢
刘芳
诸永志
徐为民
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Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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Priority to CN 201110003353 priority Critical patent/CN102134564A/en
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Abstract

The invention discloses a method for separating phospholipase A2 from animal muscles, belonging to the technical field of processing of agricultural products. The method comprises the steps of: extracting high-molecular-weight cytoplasm type pure phospholipase A2 from animal muscles or waste lean meat after processing, extracting crude enzyme firstly, and then purifying the phospholipase A2 by using an ion exchange column to obtain a phospholipase A2 of which the molecular weight is approximately 201KD. The method for separating phospholipase A2 from animal muscles has the characteristics of fast and concise purifying method and high animal source phospholipids hydrolyzing efficiency of the obtained phospholipase A2, and has broad application prospect in the fields of medicines, foods, biochemistry and the like.

Description

A kind of from animal muscle separating phospholipids enzyme A＜sub 2＜/sub method
Technical field
The invention belongs to technical field of agricultural product process, relate to a kind of from animal muscle separating phospholipids enzyme A 2Method.
Background technology
Phospholipase A 2Have the various biological function, as the conduction of lipid metabolism, signal and diminish inflammation etc., it is also relevant with myotoxin, neurotoxin and cardiotoxin etc., so phospholipase A 2Separation and purification and property research thereof be one of focus of medical field.Simultaneously because phospholipase A 2Can generate lysophospholipid (performances such as the wetting ability of lysophospholipid, emulsifying stability are better than common phosphatide) by catalytic hydrolysis glyceryl phosphatide Sn-2 position ester bond, so phospholipase A 2Also be widely used at a plurality of industrial fields (as food, medicine etc.).
At present, domestic and international researchist is to the phospholipase A in snake venom, bee venom, pig pancreas and the microorganism 2Carried out a large amount of research, wherein to phospholipase A in the snake venom 2Clone, expression and crystalline structure simulation etc. all have play-by-play, in addition to the bee venom phospholipase A 2DNA reorganization and structure also study, but these enzymes substantially all belong to lower molecular weight secretor type enzyme, and generally lower to the efficient of animal source phosphatide (as Ovum Gallus domesticus Flavus lecithin) hydrolysis.
Up to the present, can extraction phospholipase A 2Measure lessly, can not satisfy high molecular weight cell slurry matter type phospholipase A 2The needs of pure product.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can obtain phospholipase A in a large number 2Separating phospholipids enzyme A from animal muscle 2Method.
The inventive method step is:
1) gets the animal meat tissue and mince homogenate in the back adding Tris-HCl damping fluid, form slurries;
2) under 4 ℃ envrionment temperature, that slurries are centrifugal, get supernatant liquor, the ammonium sulfate that adds 70% saturation ratio then in supernatant liquor is saltoutd, and then centrifugal under 4 ℃ envrionment temperature, and throw out with the dissolving of Tris-HCl damping fluid, is obtained mixing solutions;
3) mixing solutions is placed dialysis tubing, the dialysis of desalting in the Tris-HCl damping fluid obtains the dialysate that desalts;
4) with the dialysate centrifugal removal metaprotein under 4 ℃ envrionment temperature that desalts, obtain phospholipase A 2Crude product;
5) with protein purification system purifying phospholipase A 2Crude product gets phospholipase A 2Pure product;
It is characterized in that: in the described step 5), protein purification system is: the ion exchange column that adopts source 15Q, 15 * 5.5 cm, with pH be 8.0, concentration is that 50mmol/L, flow velocity are the Tris-HCl damping fluid balance of 0.1mL/min, after treating that uv-absorbing and electricity are led baseline stability, again with pH be 8.0, the Tris-HCl damping fluid that contains 1mol/LNaCl carries out linear elution, the flow velocity that the Tris-HCl damping fluid of control NaCl enters ion exchange column is 1.5mL/min, begin to collect filtrate, stop to collect when the NaCl of filtrate concentration reaches 100%; The filtrate of collecting is carried out ultrafiltration and concentration, obtain phospholipase A 2Pure product.
The present invention adopts and extract high molecular weight cell slurry matter type phospholipase A from animal muscle or processing back depleted lean meat 2Pure product at first carry out thick enzyme extraction, use ion exchange column to phospholipase A then 2Carry out purifying.The present invention can satisfy high molecular weight cell slurry matter type phospholipase A 2The needs of pure product, simultaneously to the hydrolysis efficiency of animal source phosphatide (as Ovum Gallus domesticus Flavus lecithin) also than higher, thereby remedied phospholipase A at present commonly used 2The deficiency of product.Therefore the present invention is with a wide range of applications in fields such as medicine, food, biochemistry.
The present invention adopts ion exchange method separating phospholipids enzyme A from animal muscle 2, its advantage is:
(1) can obtain high molecular weight cell slurry matter type phospholipase A 2Pure product, common phospholipase A 2Molecular weight is 12-18KD, and the phospholipase A of present method gained 2Molecular weight is 201KD, can satisfy in the research and production such phospholipase A 2Needs.
(2) separation method is succinct fast, and protein purification only need can be finished through ion exchange column.
(3) high by 30.5% to the hydrolysis efficiency of egg phospholipids.With the Ovum Gallus domesticus Flavus lecithin is substrate, 25 ℃, and common phospholipase A 2Vigor be 97.6 μ mol substrate/hg albumen, and the phospholipase A of present method gained 2Vigor be 127.37 μ mol substrate/hg albumen.
Description of drawings
Fig. 1 is a phospholipase A 2The chromatography of ions figure of purifying.
Fig. 2 is a gained phospholipase A of the present invention 2Active electrophoresis figure.
Fig. 3 is a gained phospholipase A of the present invention 2The thin-layer chromatogram of enzymolysis product.
Fig. 4 is a gained phospholipase A of the present invention 2The gas chromatogram of enzymolysis product.
Embodiment
One, extract concrete steps:
(1) gets the duck biceps muscle of thigh, mince after rejecting visible reticular tissue and fatty tissue, take by weighing 30g, under 4 ℃ of envrionment temperatures, add 100mL, pH value and be 8.0, concentration is the Tris-HCl damping fluid of 0.1mol/L, low whipping speed is homogenate three times (each 20s) under the condition of 12000rpm/min, forms slurries.
(2) under 4 ℃ of envrionment temperatures, with the centrifugal 20min of slurries, get supernatant liquor, with four layers of filtered through gauze, the ammonium sulfate that adds 70% saturation ratio is then saltoutd.
Under 4 ℃ of envrionment temperatures, get the back product centrifugal 20min that saltouts, taking precipitate dissolves with the Tris-HCl damping fluid that 5mL, pH value are 8.0, concentration is 0.1 mol/L, obtains mixing solutions.
(3) mixing solutions is placed the dialysis tubing of MWCO=10K Da, under 4 ℃ of envrionment temperatures,, obtain the dialysate that desalts with the Tris-HCl damping fluid that pH is 8.0, concentration the is 0.1 mol/L 24h that desalts that dialyses.
(4) under 4 ℃ of envrionment temperatures, with the centrifugal 10min of the dialysate that desalts, remove metaprotein, obtain phospholipase A 2Crude product.
(5) get phospholipase A at every turn 2Crude product 1.0mL protein purification system purifying, concrete operations are as follows:
Get the ion exchange column of source 15Q, 15 * 5.5 cm, earlier with the 1.0mL phospholipase A 2Crude product adds ion exchange column, be that 0.1mL/min feeds that the pH value is 8.0, concentration is the Tris-HCl damping fluid balance of 50mmol/L with the flow velocity, after treating that uv-absorbing and electricity are led baseline stability, again with the Tris-HCl damping fluid (50mmol/L that contains 1mol/L NaCl, pH8.0) linear elution, the flow velocity that the Tris-HCl damping fluid of control NaCl enters ion exchange column is 1.5mL/min, begins to collect filtrate, stops to collect when the NaCl of filtrate concentration reaches 100%.
The filtrate of collecting is carried out ultrafiltration and concentration (MWCO=10K Da), be phospholipase A 2Pure product.
Two, identification and analysis
1, the sample of collecting is carried out ion chromatography, as shown in Figure 1, peak 7 is independent, and visible gained eluted product is that chromatography is pure.
2, sample and the standard substance of collecting are carried out electrophoretic analysis, as shown in Figure 2, the molecular weight of sample is about 201KD, and it is pure that No. 7 peaks that show acquisition have reached electrophoresis.
3, the sample of collecting is carried out the thin layer chromatography analysis of enzymolysis product, as shown in Figure 3, prove that No. 7 peaks that obtain are phospholipase A 2Activated protein.
4, the sample of collecting is carried out the gas-chromatography map analysis of enzymolysis product, as shown in Figure 4, prove that No. 7 peaks that obtain are phospholipase A 2Activated protein.

Claims (1)

1. separating phospholipids enzyme A from animal muscle 2Method, may further comprise the steps:
1) gets the animal meat tissue and mince homogenate in the back adding Tris-HCl damping fluid, form slurries;
2) under 4 ℃ envrionment temperature, that slurries are centrifugal, get supernatant liquor, the ammonium sulfate that adds 70% saturation ratio then in supernatant liquor is saltoutd, and then centrifugal under 4 ℃ envrionment temperature, and throw out with the dissolving of Tris-HCl damping fluid, is obtained mixing solutions;
3) mixing solutions is placed dialysis tubing, the dialysis of desalting in the Tris-HCl damping fluid obtains the dialysate that desalts;
4) with the dialysate centrifugal removal metaprotein under 4 ℃ envrionment temperature that desalts, obtain phospholipase A 2Crude product;
5) with protein purification system purifying phospholipase A 2Crude product gets phospholipase A 2Pure product;
It is characterized in that: in the described step 5), protein purification system is: the ion exchange column that adopts source 15Q, 15 * 5.5 cm, with pH be 8.0, concentration is that 50mmol/L, flow velocity are the Tris-HCl damping fluid balance of 0.1mL/min, after treating that uv-absorbing and electricity are led baseline stability, again with pH be 8.0, the Tris-HCl damping fluid that contains 1mol/LNaCl carries out linear elution, the flow velocity that the Tris-HCl damping fluid of control NaCl enters ion exchange column is 1.5mL/min, begin to collect filtrate, stop to collect when the NaCl of filtrate concentration reaches 100%; The filtrate of collecting is carried out ultrafiltration and concentration, obtain phospholipase A 2Pure product.
CN 201110003353 2011-01-10 2011-01-10 Method for separating phospholipase A2 from animal muscles Pending CN102134564A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1236392A (en) * 1996-09-05 1999-11-24 昂尼克斯药物公司 Phsopholipase D and DNA sequences

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1236392A (en) * 1996-09-05 1999-11-24 昂尼克斯药物公司 Phsopholipase D and DNA sequences

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
《Biochimica et Biophysica Acta》 19861231 Ruth M. Kramer等 Solubilization and properties of Ca2+-dependent human platelet phospholipase A2 第394-403页 第878卷, *
《食品工业科技》 20090630 王道营等 鸭肉磷脂酶的提取及水解模拟体系初探 , 第06期 *

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Application publication date: 20110727