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Method for purifying neutral or alkaline protease

A protease and alkaline technology, which is applied in the field of purifying neutral or alkaline protease, can solve the problems of purification efficiency, long time consumption, and many purification steps, etc., and achieve simple method, low enzyme activity loss, and few operation steps Effect

Pending Publication Date: 2019-12-10
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has many purification steps, takes a long time, and is expensive. At the same time, the purification efficiency needs to be improved. Therefore, it has become an important factor restricting the production of high-end protease preparations.

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  • Method for purifying neutral or alkaline protease
  • Method for purifying neutral or alkaline protease
  • Method for purifying neutral or alkaline protease

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preparation example Construction

[0038] 6) Preparation of the mutant affinity column and its application in the purification of neutral or alkaline protease: connect the purified recombinant protein of eglinC to the sepharose4B column material to obtain the eglinC mutant affinity column, and use the eglinC mutant affinity And column purification of neutral protease or alkaline protease.

[0039] The second scheme of this description, according to a method for purifying neutral or alkaline protease described in the first scheme, after the amino acid sequence in the step 2) is optimized by codons, chemically synthesize DNA suitable for expression in Escherichia coli sequence.

[0040] The third scheme of this description, according to a method for purifying neutral or alkaline protease described in the first scheme, the recombinant expression vector in the step 3) is: the DNA sequence of the eglinC mutant synthesized is passed through NdeI and BamHI Restriction digestion, connection to the pET3a vector after t...

Embodiment 1

[0049] Embodiment 1: a kind of method for purifying neutral or alkaline protease, comprises the following steps:

[0050] 1) Chemical synthesis of eglinC protein

[0051] According to the protein sequence of leech XD0802.1 (Hirudo medicinalis) in GeneBank, the Thr Glu Phe Gly Ser Glu Leu at positions 1-7 of the N-terminal of the eglinC protein was deleted at the same time to obtain the amino acid sequence of the eglinC mutant;

[0052] 2) Gene optimization

[0053] According to the amino acid sequence of the eglinC mutant, on the https: / / www.jcat.de / start.jsp website, optimize the DNA sequence of the eglinC mutant to facilitate expression in Escherichia coli, and add at both ends of the DNA sequence of the eglinC mutant NdeI and BamHI restriction sites.

[0054] 3) Construction of recombinant vector

[0055] The DNA sequence of the synthesized eglinC mutant was digested with Nde I and BamHI, connected to the pET3a vector after the same digestion, and the pET3a-eglin C recom...

Embodiment 2

[0063] Embodiment 2: a kind of method for purifying neutral or alkaline protease, comprises the following steps:

[0064] 1) Chemical synthesis of eglinC protein

[0065] According to the protein sequence of leech XD0802.1 (Hirudo medicinalis) in GeneBank, the Thr Glu Phe Gly Ser Glu Leu at positions 1-7 of the N-terminal of the eglinC protein was deleted at the same time to obtain the amino acid sequence of the eglinC mutant;

[0066] 2) Gene optimization

[0067]On the https: / / www.jcat.de / start.jsp website, optimize the DNA sequence of the eglinC mutant for expression in Escherichia coli, and add NdeI and BamHI restriction sites at both ends of the DNA sequence of the eglinC mutant.

[0068] 3) Construction of recombinant vector

[0069] The DNA sequence of the synthesized eglinC mutant was digested with HindIII and BamHI, and then connected to the pQE30 vector after the same digestion to construct the pQE30-eglinC recombinant expression vector.

[0070] 4) Recombinant ex...

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Abstract

The invention discloses a method for purifying neutral or alkaline protease. The method comprises the steps of chemically synthesizing a DNA sequence encoding an eglinC mutant, constructing a recombinant expression vector of the eglinC mutant, recombinantly expressing and purifying an eglinC mutant protein by utilizing escherichia coli, coupling the protein to a sepharose 4B column material, and separating and purifying the neutral or alkaline protease expressed by bacteria by utilizing the specific combination of the eglinC mutant and protease. The affinity purification system constructed bythe eglinC mutant is simple in method, few in operation steps, low in enzyme activity loss and high in purification multiple.

Description

technical field [0001] The invention relates to the biological field, in particular to a method for purifying neutral or alkaline protease. Background technique [0002] Protease is a proteolytic enzyme that catalyzes the hydrolysis of peptide bonds in proteins. Proteases are divided into different types of proteases according to different standards. According to their active centers and optimum pH values, proteases can be divided into serine proteases, sulfhydryl proteases, metalloproteases and aspartic acid proteases; according to their optimal pH values ​​for reactions , can be divided into acid protease, neutral protease and alkaline protease; of course, it can also be classified according to its source, such as papain, bromelain and so on. Protease is a very important industrial enzyme, widely used in washing industry, food, medicine, leather and other industries. Specifically, in the washing industry, it is mainly used for the production of enzyme-added washing powde...

Claims

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Application Information

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IPC IPC(8): C12N9/50
CPCC12N9/50
Inventor 石亚伟文阳宣
Owner SHANXI UNIV
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