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Deep-sea cold-adapted and salt-tolerant collagenase as well as encoding gene myr02 and application of same

A collagenase and cold-adaptive technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of less collagenase and limited application, and achieve good cold adaptability, salt tolerance, and high activity

Inactive Publication Date: 2011-09-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few related studies on microbial collagenases, and the reported bacterial collagenases are mostly accompanied by the production of toxins, which limits their application.
At the same time, due to the limitation of sampling and culture conditions, there are still few reports on collagenase from marine sources, which is worthy of further investigation.
[0004] Since collagen is a water-insoluble protein, most of it is deposited in seabed sediments in the ocean. Therefore, deep-sea sediments are good materials for isolating marine bacterial collagenase. Collagenase is still low

Method used

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  • Deep-sea cold-adapted and salt-tolerant collagenase as well as encoding gene myr02 and application of same
  • Deep-sea cold-adapted and salt-tolerant collagenase as well as encoding gene myr02 and application of same
  • Deep-sea cold-adapted and salt-tolerant collagenase as well as encoding gene myr02 and application of same

Examples

Experimental program
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Effect test

Embodiment 1

[0034]A strain of Myroides profundi D25, which was preserved in the China Center for Type Culture Collection on July 13, 2008, address: China, Wuhan, Wuhan University, strain preservation number: CCTCC NO.M 208030.

Embodiment 2

[0036] The nucleotide sequence of the collagenase gene myr02 extracted from Myroides profundi D25 is shown in SEQ ID NO.1.

[0037] The collagenase myr02 encoded by the above collagenase gene myr02 has an amino acid sequence shown in SEQ ID NO.12.

[0038] Collagenase gene myr02 contains a 2040bp open reading frame encoding collagenase myr02, the start codon is located at 1bp, the stop codon is located at 2038bp, and a total of 679 amino acids are encoded. The corresponding relationship between nucleotides and encoded amino acids in the gene is as follows: Figure 7 shown.

[0039] The preparation method of collagenase gene myr02 is as follows:

[0040] 1. Cloning of the gene encoding collagenase myr02

[0041] 1.1 For the extraction of the genomic DNA of Myroides profundi D25, refer to the instructions of the Genome Extraction Kit of Biotec Company:

[0042] (1) Take 1ml of Myroides profundi D25 bacteria liquid, centrifuge at 10000rpm for 30sec, discard the supernatant, co...

Embodiment 3

[0081] Example 3: Purification of collagenase myr02

[0082] The psychrotrophic bacteria (Myroides profundi) D25 was inoculated in the fermentation medium (artificial seawater containing 0.2% yeast powder, 1% gelatin, pH 8.5), and cultured at 15° C., 200 rpm for 84 hours. The fermentation broth was centrifuged at 10000g for 10min at 4°C. The supernatant was collected for 55% ammonium sulfate precipitation, left standing overnight, 4°C, centrifuged at 10,000g for 10min to collect the precipitate, redissolved with 50mM Tris-HCl buffer (pH 9.5) and dialyzed overnight for desalination, then centrifuged at 10,000g for 15min at 4°C. The supernatant was passed through a DEAE-Sepharose Fast Flow chromatography column at a speed of 0.4ml / min and then eluted with a gradient of 0-0.8M NaCl. The eluted active fraction was collected, concentrated and further separated and purified by supherdex G75. Purified protease was tested for purity by 7.5% SDS-PAGE ( image 3 )

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Abstract

The invention relates to a deep-sea cold-adapted and salt-tolerant collagenase as well as an encoding gene myr02 and application of the same, belonging to the field of biotechnology. The nucleotide sequence of a deep-sea cold-adapted and salt-tolerant collagenase gene myr02 is as shown in SEQ ID NO.1, and the nucleotide sequence of the collagenase myr02 encoded by the collagenase gene myr02 is as shown in SEQ ID NO.12. The collagenase myr02 can be used for degrading insoluble I type collagens, also has a higher activity for soluble substrates such as gelatins and the like, and has better cold adaptability and salt tolerance.

Description

technical field [0001] The invention relates to a deep-sea cold-adapted and salt-tolerant collagenase, its encoding gene myr02 and its application, and belongs to the technical field of biotechnology. Background technique [0002] Collagen is widely distributed in animal skin, bones, teeth, and blood vessels, and is the main component of the extracellular matrix. It has a unique triple helix structure, which is difficult to be hydrolyzed by general proteases, and can only be hydrolyzed by collagenase. Collagenase, also referred to as collagenase for short, is a proteolytic enzyme characterized by hydrolyzing insoluble collagen, which exists in multiple protease families, such as serine protease family and metalloprotease family. At present, collagenase has good application value in various aspects such as medical treatment and industry. In terms of medical treatment, collagenase can degrade the collagen fibers at the edge of the wound, which has a good debridement effect, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10
Inventor 张玉忠冉丽媛陈秀兰周明扬张熙颖何海伦解彬彬石梅周百成
Owner SHANDONG UNIV
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