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MindMap Gallery Protein extraction, separation and purification

Protein extraction, separation and purification

This is a mind map about protein extraction, separation and purification. The main contents include: precision verification, purification and separation, process optimization, extraction, sample preparation, and literature overview.

Edited at 2024-02-16 14:14:50

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Protein extraction, separation and purification

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Outline

Protein extraction, separation and purification

Literature overview

Purpose

1. Optimization of alkali solution and acid precipitation method for SPI extraction

2. Identify SPI purity

3. Provide basic data and reference for the preparation of SPI standards,

Experimental Materials

1. Experimental Materials

Soybeans, commercially available, Tianmen, Hubei; Sepharose 6B, relative molecular mass standard protein: Pharmacia Company.

2. Main reagents

β-Mercaptoethanol, acrylamide, SDS: Sigma.

3. Instruments and equipment

Automatic Kjeldahl nitrogen analyzer, 752-type spectrophotometer: Shanghai Precision Instrument Company; UV-265 UV-visible recording spectrometer: Hitachi, Japan; PHS-3C pH meter: Shanghai Leici Instrument Factory; SCIENTZ-18N freeze dryer : Ningbo Xinzhi Biotechnology Co., Ltd.

experimental method

1. Extraction Method

Alkali extraction and acid precipitation method

2. Isolation and Purification

gel chromatography

3. Content purity determination

Kjeldahl method

SDS-PAGE method

experiment procedure

1. Sample Preparation

1. Alkali extraction, acid precipitation, SPI extraction

1. Repeat five times to precipitate SPI

1. Sepharose gel filtration SPI

Experimental results

1. Extraction rate: 82.58%

2. Before purification: 92.65%

After purification: 98.56%

Remarkable purification effect

Using soybean meal after oil extraction from soybean flour as raw material, the soybean protein isolate is extracted using the acid precipitation method. Taking extraction pH, material-to-liquid ratio, extraction temperature, and extraction time as factors to examine, single factor experiments and orthogonal experiments were used to determine the optimal extraction process conditions for soybean protein isolate, which was then prepared through five times of precipitation and gel filtration. The protein was purified and its purity was confirmed by Kjeldahl method and SDS-PAGE. The results show that when the alkali solution is pH 8, the material-to-liquid ratio is 1:14g/mL, the extraction temperature is 50°C, and the extraction time is 60 minutes, the extraction rate of soybean protein isolate reaches 82.58%, and the protein content of the crude protein extract is 92.65%. The target protein spectrum of gel filtration showed a single elution peak, and the SDS-PAGE spectrum showed only a few contaminant bands. The total protein content after purification was 98.56%.

Sample Preparation

Soybean flour is passed through a 40-mesh sieve and degreased with petroleum ether to finally obtain to defatted soy flour.

extract

Alkali extraction, acid precipitation, SPI extraction

1. Mix soybean meal and water at a mass ratio of 1:15, and stir for 1 hour at room temperature and pH 8.0. Then separate the soybean meal aqueous solution slurry, centrifuge at 3000r/min for 15min, discard the precipitate, adjust the supernatant to pH 4.5, let it stand for 30min, and centrifuge again at 3000r/min for 25min to collect the precipitate.

(At pH 8.0, the alkaline environment dissolves the soybean protein in the soybean meal; at pH 4.5, the acidic condition precipitates the protein)

2. Wash with deionized water three times and then redissolve in 5 times the distilled water, adjust the pH to 7.5, centrifuge for the third time at 30000r/min for 25min, discard the supernatant, and the precipitate is the crude extraction of soybean protein isolate (SPI) things.

pH7.5: This is a relatively stable neutral environment for soy protein isolate, which is conducive to subsequent separation and purification. Repeated elution three times with deionized water is to remove impurities in the precipitate.

Process Optimization

single-factor experiment

Determine three levels and four factors

Orthogonal test

Determine optimal process conditions

The optimal process conditions are: extraction pH 8, solid-liquid ratio 1:14g/mL, extraction temperature 50°C, and extraction time 60 minutes. Under optimal conditions, the extraction rate of soy protein isolate was 82.58%, and the protein content in the extract was 92.65%

Purification and isolation

Repeat five times to precipitate SPI

After five times of precipitation of soybean SPI prepared by acid precipitation and alkali extraction, a purified protein sample was obtained and its content was measured.

Sepharose gel filtration SPI

Use agarose gel wet sample to load the column, and use 1mol/L NaCl PBS (50mmol/L KH2PO4 -Na2HPO4, pH 7.6) to equilibrate the chromatography column; prepare the sample with PBS buffer, centrifuge to remove the supernatant, and the concentration of the protein solution should be Less than 10mg/mL (repeated experiments show that the adsorption rate is high and the equilibrium time is short at this concentration). Then put it on the column, use phosphate buffer as the eluent, perform linear gradient elution, the flow rate is 1.0mL/min, 6mL/tube is collected in separate tubes, detect with a UV detector at 280nm, freeze-dry, and analyze by SDS-PAGE.

The obtained target protein spectrum showed a single elution peak, and the total protein content of the purified SPI reached 98.56%.

Precision verification

SDS-PAGE electrophoresis verification of SPI

There are only a few mixed bands in the electrophoresis pattern, indicating that the protein is of high purity.

The nitrogen content in protein is about 16%, Protein content = Nitrogen content × 6.25

Protein extraction rate = extracted soy protein content/soybean Protein content ×100%