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Gene engineering bacterium for producing keratinase escherichia coli and application thereof

A technology of genetically engineered bacteria and keratinase, applied in bacteria, hydrolase, microorganism-based methods, etc., can solve the problems of easy loss, disappearance, and reduced protein expression.

Active Publication Date: 2013-01-09
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Gene engineering bacterium for producing keratinase escherichia coli and application thereof
  • Gene engineering bacterium for producing keratinase escherichia coli and application thereof
  • Gene engineering bacterium for producing keratinase escherichia coli and application thereof

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Effect test

Embodiment 1

[0017] Construction and Identification of Embodiment 1 Recombinant Bacteria

[0018] 1) According to the gene sequence design on the NCBI website (GenBank: S78160.1), the primer sequences are as follows:

[0019] ker1-F: 5'-CATG CCATGG ATGCTCAGCCGGCGAAAAAT-3'

[0020] ker1-R: 5'-CGC CTCGAG TTATTGAGCGGCAGCTTCGA-3'

[0021] The keratinase gene was cloned using the genome of Bacillus licheniformis BBE1 (which has been deposited in the China Center for Type Culture Collection in the patent application of our unit with the patent number 201110343655.5, and the preservation number is CCTCC M 2011319) as a template.

[0022] PCR reaction system: Add the following reagents in sequence to a 0.2mL PCR tube: 5×prime STAR PCR buffer II (Mg 2+ plus) 5 μl; DNTP Mixture 4 μl; template DNA 1 μl; upstream and downstream primers 1 μl; Taq enzyme 0.5 μl; add double distilled water to a final volume of 50 μl. PCR amplification conditions: pre-denaturation at 94°C for 5min; denaturation at ...

Embodiment 2

[0027] Embodiment 2 fermentation produces keratinase

[0028] Medium: LB medium (1L) for seed and slant medium: tryptone 10g, yeast extract 5g, NaCl 10g, adjust pH to 7.0 with 1N NaOH; add agar 15g to slant medium; basic fermentation medium is TB Culture medium (1L): 900mL deionized water, 12g tryptone, 24g yeast extract, 10mL glycerol, autoclaved, cooled to 60°C, add 100mL sterilized potassium phosphate buffer;

[0029] Cultivation method: Cultivate to OD at 20°C and 200rpm 600 The seeds in 2.5 were transferred to the basic fermentation medium with an inoculum of 3%, and cultivated at 20°C and 200rpm;

[0030] Induction conditions: the induction OD value is 2.5, the recombinant bacteria are induced at 20° C. for 30 hours, and the IPTG concentration is 0.05 mM.

[0031] Using the empty vector as a control, a protein band with a molecular weight of about 37.5kDa was obtained by protein electrophoresis (SDS-PAGE) (see image 3 ), and the total enzyme activity of the recombina...

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Abstract

The invention discloses a gene engineering bacterium for producing keratinase escherichia coli and an application thereof, and belongs to the technical field of the gene engineering. The keratinase (ker) gene of bacillus licheniformis (Bacillus licheniformis BBE11-1) is connected to an escherichia coli expression vector pET22b(+) by the recombination DNA (deoxyribonucleic acid) technology; Escherichia coli BL21(DE3) is converted; one plant of combination escherichia coli BL21(Escherichia coli DE3)-pET22b(+)-ker capable of generating higher keratinase is obtained by screening and identifying; and the collection number is CCTCC (China center for type culture collection) No: M 2012180. The enzyme activity of the keratinase expressed by the bacterial strain is 256U / mL, and a good foundation is laid for the large-scale production of the keratinase.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing keratinase and an application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Keratinase is an enzyme that can specifically degrade keratin, which is produced by various microorganisms such as bacteria, actinomycetes and fungi. As a by-product of poultry breeding and slaughtering industry, feathers have a huge annual output and are rich in protein and amino acids, so they are potential excellent protein resources. Reasonable utilization of feathers can reduce the pollution of waste to the environment on the one hand, and can be used as a feed protein for livestock and poultry feeding. In the traditional process of using feathers, acid-base hydrolysis is mainly used. Thermal degradation and other methods, the former has the problem of polluting the environment, the latter consumes a lot of energy, and can destroy some amino acids, redu...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/52C12R1/19
Inventor 陈坚刘柏宏张娟堵国成
Owner JIANGNAN UNIV
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