Preparation method of recombinant halophilic archaea protease

A technology of halophilic archaea and protease, applied in the field of bioengineering, can solve the problems of low expression of extracellular enzymes, denaturation and inactivation, slow growth of halophilic archaea, etc., and achieves simple purification steps, short production cycle, good general-purpose sexual effect

Active Publication Date: 2020-03-06
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disadvantages of slow growth of halophilic archaea, high cost of culture medium and high-salt-tolerant fermentation equipment, low expression of extracellular enzymes, and cumbersome purification steps increase the production cost and production cycle of pure enzymes.
Halophilic archaeal protea

Method used

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  • Preparation method of recombinant halophilic archaea protease
  • Preparation method of recombinant halophilic archaea protease
  • Preparation method of recombinant halophilic archaea protease

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Experimental program
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Effect test

Embodiment 1

[0036] Heterologous expression of recombinant halophilic archaeal protease (HPDC1);

[0037] (1) Target gene PCR amplification

[0038] Use a sterile inoculation loop to take a ring of strain Halostella sp.DL-M4 cells (isolated from salted kelp, preserved in CGMCC 1.13603) in 30 μL of sterile water, boil for 10 minutes, centrifuge briefly and take the supernatant as a PCR template; specificity Primer forward primer F sequence is 5'-ATA CCATGG CAAATAATGGCAACCCAT-3', reverse primer R sequence is 5'-ATA CTCGAG GCCACCGCCGTTGCCGAC-3', the underlined sequences are NcoI restriction site and XhoI restriction site, respectively. The enzyme used for PCR amplification was KOD-PLUS (purchased from TOYOBO Company), and the amplification system was 50 μL.

[0039]The dosage of each ingredient is as follows:

[0040]

[0041]

[0042] PCR amplification program:

[0043]

[0044] (2) Electrophoresis verification gel cutting recovery;

[0045] Use 1% agarose gel electrophoresi...

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Abstract

The invention belongs to the field of bioengineering, and particularly relates to a preparation method of recombinant halophilic archaea protease. The method comprises the following steps: constructing a recombinant plasmid by taking a genome of Halostella sp. DL-M4 as a template and taking a pET28a plasmid as an expression vector; transforming escherichia coli cell by recombinant plasmid, inducing expression, centrifugally collecting thalli for cell resuspension and crushing, centrifuging again, taking a supernatant, adding beta-mercaptoethanol to obtain a mixed solution, enabling the mixed solution to pass through a nickel column, combining zymogen with a nickel medium for renaturation, and finally performing secondary impurity protein removal and target protein elution, performing ultrafiltration centrifugal concentration and performing gel filtration chromatography to obtain the recombinant halophilic archaea protease. The method provided by the invention has good universality, simpler purification steps and shorter production cycle, the obtained protease has high purity, and finally the halophilic archaea protease with specific enzyme activity of 1159.15 U/mg is obtained.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a preparation method of recombinant halophilic archaea protease. Background technique [0002] Protease is the most sold enzyme preparation in the world's industrial production, and it is widely used in the washing textile industry, leather industry, food processing, waste treatment, and biotechnology. In high-salt industrial production processes such as high-salt food processing and high-salt wastewater treatment, commercially available proteases are easily inactivated, which limits the use of proteases, and the halophilic archaeal proteases secreted by extreme halophilic archaea conditions to maintain high hydrolytic activity. Halophilic archaeal proteases are members of the subtilisin-like S8 family of serine proteases, and most of them have the characteristics of high salt resistance, organic solvent resistance, tolerance to various metal ions and strong stability, ...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/70
CPCC12N9/52C12N15/70
Inventor 崔恒林韩冬侯靖
Owner JIANGSU UNIV
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