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Method for extracting TAQ enzyme for amplification

An enzyme bacteria and protein liquid technology, applied in the field of active protease extraction, can solve the problems of affecting effect, active protein volatile inactivation, yield decline, etc., to ensure product yield and purity, reasonable and simple production process, and good effect. Effect

Inactive Publication Date: 2012-09-26
SHANGHAI JITAI YUANCHENG BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, because the active protein is easily denatured and inactivated, the purification is required to be carried out at low temperature (4°C). The conventional method of purifying TAQ enzyme uses low temperature cracking, repeated ion exchange, and affinity chromatography for purification. Difficult to remove completely, each chromatographic column will have a loss of about 20% during the purification process, the purity of the product can only reach more than 85%, and the yield will also decrease
Because of its purity problem, it also affects the effect of later products used for gene amplification

Method used

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  • Method for extracting TAQ enzyme for amplification
  • Method for extracting TAQ enzyme for amplification
  • Method for extracting TAQ enzyme for amplification

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Effect test

Embodiment 1

[0018] TAQ enzyme extraction

[0019] Test group: if figure 1 As shown, the fermented TAQ enzyme cell 1 was dissolved in TEBG buffer solution 2, and then the pressure crushing device 3 was used to control the pressure to 8000 psi for pressure crushing, thereby removing nucleic acids, and the temperature of the water bath device 4 was controlled to be 70°C. For the thermal stability of the protein in TEBG buffer solution, react the protein solution with nucleic acid removed in the buffer solution for 1h, the TEBG buffer solution contains a final concentration of 10% (V / V), stabilizer glycerin and glycerin to maintain the protein spatial conformation The final concentration is 0.1% (G / V), and the protective agent β-mercaptoethanol (BSA) to prevent the unsaturated bonds of proteins from being oxidized. The protein solution after the denaturation of miscellaneous proteins is chromatographically removed through the chromatographic column 5 to remove miscellaneous proteins such as ...

Embodiment 2

[0024] A kind of production technique of TAQ enzyme for extracting and amplifying by high heat method, the production technique comprises the following steps: suspend the fermented TAQ enzyme thallus with TEBG buffer solution, TEBG buffer solution contains final concentration 10% (V / V), maintain Glycerol, a stabilizer for protein spatial conformation, and a final concentration of 0.1% (G / V), and β-mercaptoethanol (BSA), a protective agent for preventing protein unsaturated bonds from being oxidized. Then control the pressure of the pressure device to 8000psi for pressure crushing, remove the protein solution after nucleic acid, use the thermal stability of the protein in the buffer, control the reaction temperature at 70°C for 1 hour, remove most of the impurity proteins, Then pass through a chromatographic column to get the desired high-purity active protein. Compared with the prior art, the production process of the TAQ enzyme for extraction and amplification by the high-hea...

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Abstract

The invention relates to a method for extracting a TAQ enzyme for amplification. The method comprises the following the steps that TAQ enzyme bacteria are suspended by a TEBG buffer solution and then are subjected to pressure fragmentation so that nucleic acids are removed and a protein solution is obtained; the protein solution undergoes a reaction in the TEBG buffer solution for 1 hour with controlling a bath temperature of 70 DEG C so that impurity proteins are removed; and the reacted protein solution is subjected to chromatography so that a high-purity active protein TAQ enzyme is obtained. Compared with the prior art, the method provided by the invention has the advantages that processes are reasonable and easy; and an active protein denatures and inactivate easily so that purification is carried out at a low temperature of 4 DEG C and the method avoids that because of repeating ion exchange and affinity chromatography adopted in the conventional purification method, a yield is reduced and a small amount of impurity proteins are difficult to be removed fully, furthest guarantees a product yield and product purity and has good later gene amplification effects.

Description

technical field [0001] The invention relates to a method for extracting active protease, in particular to a method for extracting and amplifying TAQ enzyme. Background technique [0002] At present, because the active protein is easily denatured and inactivated, the purification is required to be carried out at low temperature (4°C). The conventional method of purifying TAQ enzyme uses low temperature cracking, repeated ion exchange, and affinity chromatography for purification. Difficult to completely remove, each chromatographic column will have about 20% loss during the purification process, the purity of the product can only reach more than 85%, and the yield will also decrease. Because of its purity problem, it also affects the effect of later products used for gene amplification. Contents of the invention [0003] The object of the present invention is to provide a reasonable and simple method for extracting and amplifying TAQ enzyme with good gene amplification eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
Inventor 朱德华
Owner SHANGHAI JITAI YUANCHENG BIO TECH