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A novel metal ion tolerance keratinase and its application

A keratinase and nucleotide sequence technology, applied in the field of industrial biology, can solve the problems of unfavorable wide application and limited application of nano-silver, and achieve the effects of good tolerance, good bacteriostatic activity and simple operation.

Active Publication Date: 2020-05-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many organic solvents with high toxicity and serious pollution in these additives, which are not conducive to the wide application of the prepared nano-silver, especially limiting its application in the field of medicine (CN106180753A)

Method used

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  • A novel metal ion tolerance keratinase and its application
  • A novel metal ion tolerance keratinase and its application
  • A novel metal ion tolerance keratinase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This example illustrates a method for screening keratinase genes based on metagenomic technology.

[0043] Soil samples were collected from the place where furs were piled up in the tannery, and the total DNA of the samples was extracted with a soil genome extraction kit. According to the conserved sequence of keratinase, degenerate primers were designed for PCR amplification reaction, the amplified fragments were sequenced and compared with the NCBI database, and then primers were further designed according to the results, and the complete keratinase gene coding sequence was amplified. After the obtained keratinase sequence was analyzed by phylogenetic tree, it was found that it was most similar to the sequence of keratinase derived from Bacillus velezensis ( figure 1 ). This method is based on metagenomic technology, combined with the conserved sequence of keratinase in the database for direct amplification and screening. Compared with the screening method based on ...

Embodiment 2

[0045] This example illustrates the construction process of the keratinase Bacillus subtilis expression system.

[0046] According to the keratinase gene sequence, primers with restriction sites were designed, and the keratinase coding gene sequence was obtained by PCR amplification:

[0047] Upstream primer: 5'-CG GGATCC ATGAGAGGCAAAAAGGTATGG-3'

[0048] Downstream primer: 5'-CG ACGCGT TTACTGAGCTGCCGCCTGTA-3'

[0049] The PCR amplification reaction was carried out in a 40 μL system, and 20 μL PrimeSTAR HS, 17 μL ddH 2 O, 1 μL of template DNA, 1 μL of upstream and downstream primers. The reaction conditions were as follows: 30 cycles of denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 1 minute, and a total of 35 cycles; final extension at 72°C for 10 minutes.

[0050] The PCR product was identified by nucleic acid electrophoresis, recovered and purified by tapping gel, the recovered product w...

Embodiment 3

[0052] This example illustrates the detection method of recombinant keratinase enzyme activity.

[0053] Preparation of 1% keratin substrate: first prepare a 0.1M Tris-HCl (pH=9.0) solution, then add 5% soluble keratin mother liquor, then add deionized water to dilute the Tris-HCl solution concentration to 0.05M, And the keratin solution concentration was diluted to 1%. Enzyme reaction: Take 100 μL of moderately diluted enzyme solution, add 0.1 mL of substrate solution, place in a 50°C water bath for accurate reaction for 15 minutes, and immediately add 200 μL of 5% TCA to terminate the reaction. In the control group, 200 μL of TCA was added first, and then 0.1 mL of substrate solution was added after the enzyme reaction was completed. After the above reaction, centrifuge at 12000rpm for 5min, pipette 200μL supernatant into a new 1.5mL EP tube, add 1mL 0.4M Na 2 CO 3 , and then added 200 μL of folinol, placed in a water bath at 40°C for 20 minutes for color reaction, and th...

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Abstract

The invention relates to novel metal ion tolerance keratinase and application thereof, and provides keratinase based on metagenome technology excavation and an application method of the keratinase, belonging to the field of industrial biotechnology. The overall length of the keratinase is 1,149bp, and 382 amino acids are encoded; take B.subtilis WB600 as an example, the heterologous expression ofa keratinase gene is successfully realized. The method for obtaining the keratinase gene is convenient and feasible, the gene is subjected to recombined expression, a constructed recombination straincan realize secretory expression of recombined keratinase, the recombined keratinase has relatively good tolerance to metal ions and a surface active agent, and the stability of enzyme is relatively good. Recombinant bacteria are short in fermentation period and suitable for industrial production in large scale. In addition, the enzyme has good reducing capacity and can be applied to research in which a biological method is used for preparing nano-silver particles, and the prepared nano-silver particles have good forms and relatively good bacteriostatic activity.

Description

technical field [0001] The invention provides the gene excavation, expression and property research of a novel metal ion tolerance keratinase and its application method in the biosynthesis of nano-silver, which belongs to the field of industrial biotechnology. Background technique [0002] Keratinase is a special protease that can specifically degrade feathers, wool, hair, nails and other rich keratin. It can degrade insoluble keratin and is different from general proteases, so that keratinase can be widely used in animal husbandry, feed industry, leather industry, and pharmaceutical industry. The early research on microbial keratinase in my country mainly focused on the breeding of keratinase-producing bacteria, the separation and purification of keratinase, the study of its properties, and the application of keratinase. In 2003, Liang Bin and others from Jilin University successfully isolated the keratinase gene from Bacillus licheniformis L-25 for the first time and succ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/75C12N1/21C12P9/00C12R1/125
CPCC12N9/50C12P9/00
Inventor 史劲松龚劲松陶丽妍许正宏李恒苏畅
Owner JIANGNAN UNIV
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