Genetically engineered strain capable of producing keratinase and application thereof

A technology of genetically engineered bacteria and keratinase, applied in the direction of genetic engineering, application, plant genetic improvement, etc., can solve the problems of easy loss, restricting the growth of host bacteria, and toxicity of host bacteria.

Inactive Publication Date: 2012-10-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are also many problems to be solved in expression in heterologous hosts, such as the expression of proteins in the form of inclusion bodies; this heterologously expressed protein can sometimes be toxic to the host bacteria, limiting the growth of the host bacteria, and, in the accumulation of expression During the process, this alkaline protease will also cause its own degradation; the constructed recombinant plasmid is sometimes very unstable, and it is easy to lose during the expression process, which will cause the decrease or disappearance of protein expression

Method used

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  • Genetically engineered strain capable of producing keratinase and application thereof
  • Genetically engineered strain capable of producing keratinase and application thereof
  • Genetically engineered strain capable of producing keratinase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction and identification of recombinant bacteria

[0024] 1) The primers are designed according to the gene sequence of NCBI website (GenBank: S78160.1), and the primer sequence is as follows:

[0025] ker1-F: 5’-GGAATTC CATATG ATGAGGAAAAAGAGTTTTTGG-3’

[0026] ker1-R: 5’CGC GGATCC TTATTGAGCGGCAGCTTCG-3’

[0027] Bacillus licheniformis BBE1 has been deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC M 2011319.

[0028] PCR reaction system: Add the following reagents in order to the 0.2mL PCR tube: 5×prime STAR PCR buffer II (Mg 2+ plus) 5μl; DNTP Mixture 4μl; template DNA 1μl; upstream and downstream primers 1μl each; Taq enzyme 0.5μl; add double distilled water to a final volume of 50μl. PCR amplification conditions: 94°C pre-denaturation for 5min; 94°C denaturation for 30s, 61°C annealing for 15s, 72°C extension for 1min 20s (30 cycles); 72°C for 10min extension.

[0029] 2) Verify by 1% agarose gel electrophoresis and r...

Embodiment 2

[0033] Example 2 Enzyme activity determination and protein electrophoresis of recombinant bacteria

[0034] Medium: Seed and slant medium is LB medium (1L): tryptone 10g, yeast extract 5g, NaCl10g; slant medium with 15g agar; basic fermentation medium is LB medium with glucose (1L): Tryptone 10g, yeast extract 5g, NaCl 10g, glucose 10g;

[0035] Cultivation method: The seeds cultured overnight at 37°C and 200 rpm are transferred to the basic fermentation medium at an inoculum of 3%, and cultivated at 37°C and 200 rpm;

[0036] With the empty vector as a control, a protein band with a molecular weight of about 40kDa was obtained by protein electrophoresis (SDS-PAGE) (see image 3 ), and the enzyme activity of the recombinant bacteria was measured at the same time. The enzyme activity was 522U / mL, which was nearly 2 times higher than the total enzyme activity of the wild bacteria (320U / mL).

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Abstract

The invention discloses a genetically engineered strain capable of producing keratinase and application thereof and belongs to the technical field of genetic engineering. Gene of keratinase from Bacillus licheniformis BBE11-1 is cloned and connected to a bacillus subtilis expression vector pMA 0911 by DNA recombination technology and is converted into bacillus subtilis WB600. The recombinant bacillus subtilis strain WB600-pMA 0911-ker capable of producing a high amount of keratinase is obtained by screening and identifying. The recombinant bacillus subtilis strain WB600-pMA 0911-ker is preserved in China center for type culture collection under the number of CCTCC NO.M 2012066 on March 8 2012. Activity of the keratinase expressed by the genetically engineered strain is 522U/mL about two times of total enzyme activity of wild mushrooms. Therefore, solid foundation for large-scale production of the keratinase is laid.

Description

Technical field [0001] The invention relates to a genetically engineered bacterium producing keratinase and its application, and belongs to the technical field of genetic engineering. Background technique [0002] Keratinase is an enzyme that can specifically degrade keratin. It is produced by bacteria, actinomycetes and fungi. Feather, as a by-product of the poultry breeding and slaughtering industry, has huge annual output, and is rich in protein and amino acids, which is a potential excellent protein resource. On the one hand, the rational use of feathers can reduce the pollution of waste to the environment, and at the same time, it can be used as a feed protein for livestock and poultry breeding. In the traditional process of using feathers, acid-base hydrolysis is mainly used. Thermal degradation and other methods, the former has environmental pollution problems, and the latter consumes more energy, and can destroy some amino acids, reducing the nutritional value of the pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/56C12N15/57C12N15/75A23K1/16C12R1/125
Inventor 陈坚刘柏宏张娟堵国成
Owner JIANGNAN UNIV
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