A method for separating and purifying soybean protease inhibitor
A technology of soybean protease and inhibitor, which is applied in the field of protein separation and purification, can solve the problems of complicated operation and poor purification effect, and achieve the effects of controllable operation, low loss rate and mild environment
Active Publication Date: 2020-07-28
CHINA AGRI UNIV
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Problems solved by technology
This method overcomes the disadvantages of complex operation and poor purification effect of the existing separation and purification technology, and establishes a preliminary extraction method using isoelectric point precipitation, and further uses the technology of weak anion gel column combined with high-efficiency molecular sieve chromatographic column to extract from raw soybeans High-purity Kunitz-type trypsin inhibitor method, and optimize the conditions in all purification processes, the loss rate is less than 10%, and the product purity is more than 95%
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Embodiment 1
[0039] Example 1 Preparation of high-purity soybean Kunitz-type trypsin inhibitor
[0040] This embodiment takes the purification of Kunitz-type trypsin inhibitor in raw soybean as an example to illustrate the method of the present invention.
[0041] 1. Prepare the required solution
[0042] (1) 20mM Bis-Tris-HCl buffer solution with pH 6.4 is prepared as follows: 209.2g Bis-Tris base, add 800mL pure water, after dissolving, adjust the pH value to 6.4 with concentrated hydrochloric acid, and then use pure water to make up the volume again To 1L, make 1M stock solution, and then dilute with pure water at a volume ratio of 1:49 to 20mM Bis-Tris-HCl buffer solution with pH 6.4.
[0043] (2) 20mM pH6.4 Bis-Tris-HCl buffer solution containing 1M NaCl was prepared as follows: Measure 800mL of the above-prepared 20mM pH6.4 Bis-Tris-HCl buffer solution, add 58.44g NaCl to it, dissolve Afterwards, dilute to 1L with 20mM Bis-Tris-HCl buffer solution pH6.4.
[0044] (3) 50mM pH7.5PB ...
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Abstract
The invention relates to separation and purification of proteins and particularly discloses a method for separating and purifying a soybean protease inhibiting factor. Defatted soybean meal is subjected to crude extraction, gel column pre-purification and molecular sieve chromatography separation and purification successively to obtain the soybean protease inhibiting factor, wherein the crude extraction is that the defatted soybean meal and 20 mM pH6. 4Bis-Tris-HCl buffer solution are mixed according to the ratio of 1 g to 20-40 mL, room temperature extraction is performed for 2-3 hours, and then centrifugal supernatant collection is performed under the temperature condition of 3-8 DEG C to obtain a sample extraction solution. The steps of the method are simple, operation is controllable, the environment is mild, the method is good in repeatability, the loss ratio of the whole purification process is lower than 10%, the product purity is 95% or above, the soybean protease inhibiting factor can serve as an analytical standard product for detecting the Kunitz trypsin inhibiting factor content in soybean and its processed samples and can be also added to animal feed for research of an anti-nutrition mechanism.
Description
technical field [0001] The invention relates to the separation and purification of protein, in particular to the separation and purification of soybean protease inhibitor. Background technique [0002] Soybean protease inhibitor is one of the main antinutritional factors in soybean. 40% of the antinutritional effects of raw soybeans are caused by protease inhibitors. Such anti-nutritional factors can inhibit the proteolytic enzymes secreted by the pancreas in the intestinal tract of humans and animals, such as trypsin, chymotrypsin and elastase, etc., causing growth stagnation, pancreatic hyperplasia and hypertrophy, etc. Animals eat food containing protease inhibitors The diet will reduce the feed intake and daily gain, and the feed conversion rate will be reduced. There are more than 10 kinds of protease inhibitors in plants, among which Kunitz-type trypsin inhibitors (molecular weight between 22-24KD) are research hotspots at home and abroad. [0003] So far, many scho...
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IPC IPC(8): C07K14/81C07K1/36C07K1/30C07K1/18C07K1/16
CPCC07K14/8114
Inventor 贺平丽周天骄李婷婷李溱
Owner CHINA AGRI UNIV