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Method for immobilizing saccharifying enzyme

A technology of glucoamylase and cross-linking enzyme, which is applied in the field of bioengineering, can solve the problems of complex preparation process and high cost, and achieve the effects of simple operation, improved particle hardness and activity, and good thermal stability

Inactive Publication Date: 2011-08-17
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the disadvantages of high cost and complicated preparation process of previous cross-linking immobilization methods, and provide a method for immobilizing glucoamylase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Add the crude enzyme of glucoamylase to phosphate buffer (100mM, pH7.0) of 1 / 3 of the volume of the enzyme solution to dissolve and stir to obtain a crude glucoamylase solution (or centrifuge the fermentation broth at 10000rpm for 15 minutes to remove the bacteria, take the supernatant);

[0019] Slowly add solid ammonium sulfate to the enzyme solution, so that the mass percentage of ammonium sulfate in the total liquid is 80%, stir at 300rpm for 30min, and take the supernatant and centrifuge to determine the protein content every 10 minutes while stirring, after more than 95% of the enzyme is precipitated , centrifuged at 10000 rpm at 4° C. for 15 minutes to obtain a crude enzyme product, and added an equal volume of phosphate buffer (pH 7.0, 100 mM) to dissolve the crude enzyme to form a purified crude enzyme solution.

[0020] Take 1.6ml of crude enzyme solution and add 1 / 4 volume of phosphoric acid buffer (100mM, PH 7.0), then add solid ammonium sulfate, so that the...

Embodiment 2

[0024] Add the crude enzyme of glucoamylase to phosphate buffer (100mM, pH7.0) of 1 / 3 of the volume of the enzyme solution to dissolve and stir to obtain a crude glucoamylase solution (or centrifuge the fermentation broth at 10000rpm for 15 minutes to remove the bacteria, take the supernatant);

[0025] Slowly add solid ammonium sulfate to the enzyme solution, so that the mass percentage of solid ammonium sulfate in the total liquid is 80%, stir at room temperature at 300rpm for 30min, and take the supernatant and centrifuge to determine the protein content every 10 minutes while stirring, so that more than 95% of the enzyme After precipitation, centrifuge at 10,000 rpm at 4° C. for 15 minutes to obtain a crude enzyme product, and add an equal volume of phosphate buffer (pH 7.0, 100 mM) to dissolve the crude enzyme to form a purified crude enzyme solution.

[0026] Take 1.6ml of crude enzyme solution, add 1 / 4 volume of phosphoric acid buffer (100mM, PH 7.0) and then add solid ...

Embodiment 3

[0030] Add the crude enzyme of glucoamylase to phosphate buffer (100mM, pH7.0) of 1 / 3 of the volume of the enzyme solution to dissolve and stir to obtain a crude glucoamylase solution (or centrifuge the fermentation broth at 10000rpm for 15 minutes to remove the bacteria, take the supernatant);

[0031]Slowly add solid ammonium sulfate to the enzyme solution, so that the mass percentage of solid ammonium sulfate in the total liquid is 80%, stir at room temperature at 300rpm for 30min, and take the supernatant and centrifuge to determine the protein content every 10 minutes while stirring, so that more than 95% of the enzyme After precipitation, centrifuge at 10,000 rpm at 4° C. for 15 minutes to obtain a crude enzyme product, and add an equal volume of phosphate buffer (pH 7.0, 100 mM) to dissolve the crude enzyme to form a purified crude enzyme solution.

[0032] Take 1.6ml of crude enzyme solution, add 1 / 4 volume of phosphoric acid buffer and then add solid ammonium sulfate,...

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Abstract

The invention relates to a method for immobilizing saccharifying enzyme and belongs to the technical field of bioengineering. The method is improved on a cross-linked enzyme aggregate immobilization method and the improvements of the method include: a, an enzyme substrate protector is used for protection; b, room-temperature crosslinking is adopted, and glutaraldehyde is used as a cross-linking agent; and c, a low-temperature static crosslinking process is adopted. Compared with other immobilization methods, the method for immobilizing the saccharifying enzyme has the advantages that: the operation is simpler; the dextrin hydrolysis capacity of a cross-linked saccharifying enzyme aggregate compares with that of primary enzyme solution; the external environment-resistance stability is much higher than free enzyme solution; and the saccharifying enzyme can be used repeatedly for more than 2 months. Improvement and optimization both improve the hardness activity of the crosslinked enzyme particles greatly, and recycling is promoted. The method has the advantages that: the operation is simple; the cost is low; the thermal stability and storage stability of the immobilized saccharifying enzyme are high; the immobilized saccharifying enzyme has broad-spectrum pH adaptability; and the activity of the immobilized saccharifying enzyme is more than 60 percent of the activity of free enzyme.

Description

Technical field: [0001] The invention relates to a method for immobilizing glucoamylase, which belongs to the technical field of bioengineering. Background technique: [0002] Cross-linked enzyme aggregates (CLEAs) were proposed by the Sheldon group of Delfe University in the Netherlands in 2000. They mainly use inducers to physically aggregate enzymes, but their active sites are not destroyed. The protein forms a supramolecular structure, and the substrate passes through aggregation. The middle gap of the body interacts with the active site of the enzyme, so that the active site of the enzyme avoids direct interaction with the external microenvironment. At present, domestic research on CLEAs is less, while foreign research is more. [0003] The immobilization method of cross-linked enzyme aggregates has the following characteristics: a) The purity of the enzyme is not high, and complex steps such as crystallization are not required. In theory, any enzyme or protein that ca...

Claims

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Application Information

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IPC IPC(8): C12N11/00
Inventor 乔敏李小冬余泽芬张克勤周薇贾东晨
Owner YUNNAN UNIV
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