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Method for renaturing and purifying recombinant extremely heat-resistant alpha-amylase

An amylase, weight-to-volume ratio technology, applied in the biological field, can solve the problem of high operating conditions

Inactive Publication Date: 2013-07-10
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem of the alkali denaturation-heating-dilution refolding method is that it requires high operating conditions and has strict requirements on temperature and pH.

Method used

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  • Method for renaturing and purifying recombinant extremely heat-resistant alpha-amylase
  • Method for renaturing and purifying recombinant extremely heat-resistant alpha-amylase
  • Method for renaturing and purifying recombinant extremely heat-resistant alpha-amylase

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preparation example Construction

[0047] Therefore, the present invention provides a kind of preparation method of thermophilic α-amylase, it comprises the steps: (1) dissolve the inclusion body of thermophilic α-amylase with detergent solution, thereby obtain the inclusion body containing thermophilic α-amylase solution; and (2) isolating said thermophilic alpha-amylase from the solution obtained in (1).

[0048] The thermophilic α-amylase that can be used in the present invention can be from the thermophilic α-amylase of thermophilic archaea (also translated as Pyrococcus furiosus), the thermophilic α-amylase of Pyrococcus genus, Pyrococcus worriii ( Pyrococcus woesei) and thermophilic α-amylases from other thermophilic microorganisms. In the present invention, the thermophilic α-amylase may also be a variant of the thermophilic α-amylase. Representative thermophilic alpha-amylases include, but are not limited to, those encoded by the following genes: GenBank Accession Nos. U96622, AF001268, AF177906.1, AE0...

Embodiment 1

[0093] The influence of embodiment 1 denaturing agent and organic solvent on PFA amylase activity

[0094] As previously reported PFA has strong stress resistance. The present inventor has studied the influence of several denaturants and organic solvents on the activity of PFA enzyme. PFA amylase was placed in 6M urea, 4M guanidine hydrochloride, 0.2% SDS, and 1 / 8 (V / V) methanol, acetone, and ethanol to measure its enzyme activity. It was found that 4M guanidine hydrochloride would reduce the enzyme activity. Three organic solvents Little effect on the enzyme activity of PFA. However, SDS not only has no effect on the enzyme activity of PFA, but also enhances it to a certain extent. The results are shown in figure 1 .

Embodiment 2

[0095] Embodiment 2 Solubility of inclusion bodies in different concentrations of detergents

[0096] SDS has been used for the solubilization of poorly soluble proteins. According to the aforementioned verification, the detergent will not inhibit the amylase activity of PFA, so the inventors tried to use the detergent to dissolve the PFA inclusion body in order to obtain a simpler and more efficient purification method.

[0097] By measuring the solubility of several concentrations of inclusion bodies in different concentrations of detergents, it is found that as the concentration of inclusion body protein increases, the concentration of detergent required to fully dissolve inclusion bodies increases, see figure 2 .

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Abstract

The invention discloses a method for preparing a thermophilic alpha-amylase, which comprises the following steps: dissolving an inclusion body of the thermophilic alpha-amylase by using detergent solution so as to obtain solution containing the thermophilic alpha-amylase; and separating out the thermophilic alpha-amylase from the obtained solution. The thermophilic alpha-amylase obtained by the method has consistent enzymatic activity and heat resistance of native proteins. The method has the characteristics of simplicity, high efficiency, low cost and easy amplification, and is particularly suitable for the mass preparation of recombinant thermophilic alpha-amylase. Simultaneously, the method is also suitable for the renaturation and the purification of other thermophilic proteins expressed in forms of insoluble inclusion bodies.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a recombinant expression and preparation method of thermophilic α-amylase. Background technique [0002] α-amylase is one of the most commonly used enzyme preparations in industry, and is widely used in starch processing in food, textile, chemical industry and fermentation industry. The search and development of high heat-tolerant amylases has long been a key and hot topic in the field of industrial enzyme research. For amylase heat resistance, any improvement in production yield and enzyme activity has very important theoretical significance and direct commercial value. [0003] A variety of thermophilic alpha-amylases have been discovered so far. For example, alpha-amylase PFA from Pyrococcus furiosus; thermophilic alpha-amylase from Pyrococcus, thermophilic alpha-amylase from Pyrococcus woesei, etc. . P furiosus is an absolute anaerobic bacterium...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/28
Inventor 张毅王丽萨陆坚峰杨胜利
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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