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Method for fixing alpha-amylase

A technology of amylase and starch, which is applied in the field of bioengineering, can solve the problems of high cost and complicated preparation process, and achieve the effects of increased particle hardness and activity, simple operation and low cost

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 shortcomings of high cost and complicated preparation process of previous cross-linking immobilization methods, and provide a method for immobilizing α-amylase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Add the crude enzyme of α-amylase to the phosphate buffer (100mM, PH7.0) of 1 / 3 relative to the volume of the enzyme solution to dissolve 300rpm and stir at room temperature for 15min to prepare a crude α-amylase solution (or centrifuge the fermentation broth at 10000rpm 15 minutes, remove the bacteria, get the supernatant);

[0019] Slowly add solid ammonium sulfate to the enzyme solution so that the mass percentage of ammonium sulfate in the solution is 80%, stir at room temperature at 300rpm for 30min, take the supernatant and centrifuge to measure the protein content every 10 minutes while stirring, and after more than 95% of the enzyme is precipitated, Centrifuge at 10,000 rpm at 4°C for 15 min to discard the supernatant to obtain a crude enzyme product, and dissolve the crude enzyme with an equal volume of phosphate buffer (pH 7.0, 100 mM) to form a purified crude enzyme solution.

[0020] Get crude enzyme solution 1.6ml and add 1 / 4 volume of phosphate buffer (pH ...

Embodiment 2

[0024] Add the crude enzyme of α-amylase to the phosphate buffer (100mM, PH7.0) of 1 / 3 relative to the volume of the enzyme solution to dissolve 300rpm and stir at room temperature for 15min to prepare a crude α-amylase solution (or centrifuge the fermentation broth at 10000rpm 15 minutes, remove the bacteria, get the supernatant);

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

[0026] Get crude enzyme liqui...

Embodiment 3

[0030] Add the crude enzyme of α-amylase to the phosphate buffer (100mM, PH7.0) of 1 / 3 relative to the volume of the enzyme solution to dissolve 300rpm and stir at room temperature for 15min to prepare a crude α-amylase solution (or centrifuge the fermentation broth at 10000rpm 15 minutes, remove the bacteria, get the supernatant);

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

[0032] Get 1.6ml of crude enz...

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PUM

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Abstract

The invention relates to a method for fixing alpha-amylase, belonging to the technical field of gene engineering. The method for fixing alpha-amylase is an improvement based on a method for fixing a cross-linking enzyme aggregate. The improvement is that: a, an enzyme substrate protecting agent is used for protecting; b, room temperature crosslinking is performed, and glutaric dialdehyde is taken as a crosslinking agent; and c, a low-temperature standing and crosslinking process is performed. Compared with other fixing methods, the invention has the advantages: the method is simple to operate, and a crosslinking alpha-amylase aggregate has equivalent starch hydrolyzing capability to that of a protoenzyme fluid, environment resistance stability far higher than that of a free enzyme fluid and long service life, and can be used repeatedly. Through improvements and optimization, the particle hardness activity of cross-linking enzyme is greatly enhanced, and recycling is facilitated. The invention has the advantages of simpleness for operation, low cost, high thermal stability, high storage stability, broad-spectrum pH adaptability and capability of enabling the enzyme activity after fixation to reach over 96 percent of the activity of free enzymes.

Description

Technical field: [0001] The invention relates to a method for immobilizing alpha-amylase, 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 c...

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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