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Immobilization myrosin and method for producing the same

A technology of myrosinase and seeds, which is applied in the field of biochemistry and can solve problems such as difficulty in immobilization

Inactive Publication Date: 2008-05-07
ZHEJIANG GONGSHANG UNIVERSITY
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Problems solved by technology

It is more difficult to immobilize

Method used

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Embodiment Construction

[0019] Below by taking the seed of cruciferous broccoli as example, further illustrate the present invention, its specific implementation technological process is as follows:

[0020] After 200g of broccoli seeds are crushed into powder, add 600ml of buffer solution, which is a phosphate buffer solution with a pH of 6.0. The preparation method is: 300ml 0.1mol / L potassium dihydrogen phosphate+33.6ml 0.1mol / L NaOH, containing 5% glycerol , 1 mM EDTA, 3 mM DTT. After stirring, filter with gauze, centrifuge the filtrate at 12000×g for 20min at 4°C, collect the supernatant, add solid ammonium sulfate to reach 80% saturation, let stand at 4°C for 2h, then centrifuge at 12000×g for 20min at 4°C , collect the precipitate, dissolve it with a small amount of distilled water, and dialyze it with distilled water for 2 hours at 4° C. (molecular weight cut-off: 14000 D). The liquid in the dialysis bag is myrosinase solution. Take 100ml of enzyme solution and add water to dilute to 500ml, ...

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Abstract

The invention relates to immobilized myrosinase and the preparation method thereof. Cruciferae vegetable seeds, especially broccoli seeds are adopted, and glycerin, EDTA and DTT are added in the process of extracting the myrosinase to protect the enzyme activity; operation is performed at a lower temperature of 4 DEG C, to complete impurity removal and to obtain primary enzyme extracting solution; sodium alginate is added in the immobilization step, is dissolved and mixed at a temperature at which enzyme activity is not influenced, calcium chloride solution is instilled to be produced into calcium alginate granules, glutaraldehyde solution is used for treating the granules, in order to immobilize the free myrosinase. The immobilization method of the invention enables the activity of the myrosinase after being immobilized to achieve 70.84 percent; the pH value stability range of catalytic reaction is widened from 5-7 to 4-9, and the heat-resistant capacity is improved from original 55 to 65 DEG C; the frequency of use of the myrosinase is increased from the original once to three times.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to an immobilized enzyme and its preparation, in particular to a myrosinase isolated from plants and directly immobilized, and a preparation method thereof. Background technique [0002] Myrosinase is mainly found in cruciferous plants. Under certain conditions, myrosinase can decompose glucosinolate to produce glucose, sulfate, isothiocyanate, acetonitrile, thiocyanate and other substances. Many of these isothiocyanates are biologically active and have relatively good anticancer effects. [0003] At present, there are many reports on the extraction and isolation of myrosinase from different cruciferous plants in the world, but the free myrosinase has a short survival time and is difficult to reuse in enzymatic catalytic reactions, because the enzyme contains 6 pairs of disulfide bonds, the enzyme is easily damaged by heavy metal ions in the purification process, and is very se...

Claims

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Application Information

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IPC IPC(8): C12N11/04C12N9/14
Inventor 王向阳施青红沈莲清
Owner ZHEJIANG GONGSHANG UNIVERSITY
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