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Method for separating and purifying protease capable of degrading soybean protein allergens

A soybean protein, separation and purification technology, applied in the field of protein purification, can solve the problems of few enzymes, separation and purification, etc., and achieves the effects of good stability, mild conditions and easy control of the method.

Inactive Publication Date: 2015-04-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The enzymes used in the current research on the degradation of soybean protein allergens by enzymatic hydrolysis are all concentrated on commercial enzymes, and there are few studies on the separation and purification of the strains screened for the degradation of soybean allergens and their enzyme production
Although a strain of wild bacteria capable of degrading soybean allergens has been screened from the point of view of degrading soybean allergens, the enzyme capable of degrading soybean allergens has not been isolated and purified from the product of the strain, so the understanding of this enzyme is still limited. is very few

Method used

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  • Method for separating and purifying protease capable of degrading soybean protein allergens
  • Method for separating and purifying protease capable of degrading soybean protein allergens
  • Method for separating and purifying protease capable of degrading soybean protein allergens

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 protease activity assay

[0032] 1. L-tyrosine standard curve drawing: L-tyrosine standard solution is prepared according to Table 1

[0033] Table 1

[0034]

[0035]Take 1.00mL of each of the above solutions (parallel tests are required), add 5.0mL of 0.4mol / l sodium carbonate solution, and 1.00mL of Folin’s reagent solution, place in a water bath at 50±0.2°C for 20 minutes to develop color, and use a spectrophotometer At a wavelength of 660nm, in a 5mm cuvette, take the 0 tube without tyrosine as the blank, measure the absorbance respectively, and draw the standard curve with the absorbance A as the ordinate and the concentration of tyrosine as the abscissa.

[0036] 2. Determination of protease activity

[0037] Take 4 test tubes and number them 1, 2, 3, 4. Test tube No. 1 is the control. 2% casein was dissolved in PBS (sodium dihydrogen phosphate-disodium hydrogen phosphate) at pH 8.0, and then preheated at 50°C. First, add the preheated enzyme...

Embodiment 2

[0038] The purification of embodiment 2 protease

[0039] The fermentation broth was centrifuged at 7000rpm at 4°C for 20min to obtain the fermentation supernatant, and ammonium sulfate powder was added slowly at 4°C until the saturation of ammonium sulfate in the fermentation supernatant reached 50%, and the supernatant was collected after centrifugation at 7000rpm at 4°C for 20min, and the Ammonium sulfate was used to make the final concentration reach 80% saturation, and after standing at 4° C. for 1 h, the precipitate was collected by centrifugation at 4° C. at 7000 rpm. And the precipitated protein was dissolved in Tris-HCl buffer containing 25mmol / L, pH 9.0. Purified by anion exchange chromatography, the elution conditions are: equilibrium solution: 25mmol / L Tris-HCl buffer, pH 9.0; eluent: equilibrium solution containing 1mol / L NaCl; linear elution, flow rate is 1mL / min, Collect the protein eluted by about 15% of the eluent as the target protease solution, and then use...

Embodiment 3

[0042] Enzymatic properties of embodiment 3 protease

[0043] The enzymatic properties of the purified protease were studied, and it was found that the stability of the purified protease was better at pH 7.0-9.0 and 30-40°C. More than 80% of the enzyme activity; the pure enzyme is incubated at 30-40°C for 60 minutes, the stability is good, and the enzyme activity can still maintain more than 80%. Ca 2+ In the presence of 2+ , Fe 2+ and Cu 2+ It has obvious inhibitory effect on this protease; phenylmethylsulfonyl fluoride (PMSF) has a more significant inhibitory effect on this protease, but dithiodinitrobenzoic acid (DNTB) has no inhibitory effect on this protease, so it is speculated that it is a serine protease. K of the protease m 4.19g / L, V max 4.04g / (L·s), K cat 107.73s -1 , K cat / K m It is 25.71L / (g·s).

[0044] 1 Optimum reaction pH and temperature of enzyme

[0045] (1) Optimum reaction pH: the purified proteases were tested for their enzyme activity in dif...

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Abstract

The invention discloses a method for separating and purifying a protease capable of degrading soybean protein allergens, belonging to the technical field of protein purification. The method comprises the following step: separating and purifying the protease which is sourced from a wild strain Bacillus sp.BBE201108 and has the effect of degrading soybean protein allergens by sequentially adopting ammonium sulfate distributed precipitation, dialysis, anion exchange chromatography (Q FF) and gel filtration chromatography (Superdex 75). The protease disclosed by the invention can be successfully prepared by virtue of separation and purification, and a foundation for analyzing enzymatic properties and amino acid sequences of the protease and achieving heterologous expression research of the protease can be provided.

Description

technical field [0001] The invention relates to a method for separating and purifying protease capable of degrading soybean protein allergens, and belongs to the technical field of protein purification. Background technique [0002] Soybean is rich in essential amino acid components. It is a high-quality vegetable protein source for humans and livestock and poultry. It has high nutritional value. In addition, soybean protein also has excellent processing properties such as emulsification and foaming properties, as well as good Physiological functions, can lower cholesterol, reduce the risk of heart disease, etc. With the improvement of soybean protein separation technology, soybean protein has been widely used as a nutritional and functional ingredient, and processed foods based on soybean protein are currently the fastest growing category in the food industry. However, soybean protein is also one of the eight main allergens, and nearly 0.3%-0.4% of the world's population i...

Claims

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Application Information

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IPC IPC(8): C12N9/54A23J3/34A23J3/16
CPCC12N9/2417A23J3/16A23J3/346
Inventor 张娟陈坚付欧
Owner JIANGNAN UNIV
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