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Deep sea elastase gene as well as preparation method and application thereof

A technology for elastase and strain preservation, applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems that there is no M23 family elastase, achieve great application potential and ensure safety sexual effect

Inactive Publication Date: 2012-07-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulties of sampling and culturing, marine-derived elastases are rarely studied at present, and there is no report of M23 family elastases derived from deep sea.

Method used

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  • Deep sea elastase gene as well as preparation method and application thereof
  • Deep sea elastase gene as well as preparation method and application thereof
  • Deep sea elastase gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Cloning of the gene encoding cold-adapted elastase

[0038] 1. Extraction of genomic DNA of Pseudoalteromonas sp. CF6-2.

[0039] The extraction method is carried out according to the following steps and according to the instructions of the Genome Extraction Kit of Biotec Company:

[0040] (1) Take 1ml of Pseudoalteromonas sp. CF6-2, centrifuge at 10000rpm for 30sec, discard the supernatant, and collect the bacteria;

[0041](2) Add 200 μl of buffer KB to resuspend and wash the cells, centrifuge at 10,000 rpm for 30 sec, discard the supernatant and resuspend the cells in 200 μl of buffer KB by shaking or pipetting;

[0042] (3) Add 200 μl of binding solution CB, mix well, then add 20 μl proteinase K (20 mg / ml) solution, mix well, and place at 70°C for 10 minutes;

[0043] (4) After cooling, add 100 μl of isopropanol and mix thoroughly to obtain a mixed solution;

[0044] (5) Add the mixed solution prepared in step (4) into the adsorption column AC, centrif...

Embodiment 2

[0082] Example 2: Purification of cold-adapted elastase secreted by psychrotroph CF6-2

[0083] Pyrotrophic bacteria CF6-2 was inoculated in the fermentation medium (artificial seawater (prepared according to the seawater preparation method of Qingdao Haizhishui Aquarium Technology Co., Ltd.) containing 0.2wt% yeast powder and 0.3wt% elastin (purchased from sigma company) , 0.5mM CaCl 2 and 0.5mM Na 2 HPO 4 , pH 8.0), cultivated at 15°C for 48h. The fermentation broth was centrifuged at 10000 g for 10 min at 5 °C. The supernatant was dialyzed overnight with 50 mM Tris-HCl buffer (pH 9.5) and centrifuged at 10000 g for 15 min at 5°C. The supernatant was passed through the DEAE-Sepharose Fast Flow chromatography column at a speed of 2.5 min / ml, and then gradient eluted with 0-0.3M NaCl. The protease under elution detects purity with 12.5% ​​SDS-PAGE (results such as image 3 shown).

Embodiment 3

[0084] Example 3: Determination of properties of cold-adapted elastase

[0085] 1. The ability of cold-adapted elastase to lyse different types of bacteria

[0086] After the test cells were centrifuged at 10000 rpm for 1 min, the supernatant was discarded, and the cells were washed with 50 mM Tris-HCl buffer solution (pH 9.0). The washed cells were resuspended in 50 mM Tris-HCl buffer (pH 9.0) and boiled for 20 min. After cooking, dilute the sample with the same buffer solution until the absorbance at 595nm is 0.8, take 180μl of bacterial solution and add 20μl of cold-adapted elastase enzyme solution with a concentration of 10μg / ml, mix well, keep it at 25°C for half an hour, and measure the absorbance at 595nm Value changes, the control sample was replaced by Tris-HCl buffer solution. Absorbance value reduction rate was used as relative bacteriostasis activity (results are shown in Table 1).

[0087] Table 1

[0088]

[0089] 2. Analysis of the cleavage site of cold-a...

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Abstract

The invention relates to a deep sea elastase gene as well as a preparation method and an application thereof, belonging to the field of biotechnology. The invention relates to a cryophilic bacterium CF6-2, a cryophilic elastase gene extracted from the cryophilic bacterium CF6-2 and deep sea elastase expressed and translated by the gene. The cryophilic elastase derives from a deep sea low-temperature high-salinity environment. Compared with other kinds of elastase, the cryophilic elastase has higher elastin degradation efficiency in sea water at 0-30 DEG C and can exert functions at normal temperature. The cryophilic elastase is extremely unstable at the temperature above 40 DEG C and is inactivated at middle temperature, so that the safety of the cryophilic elastase is ensured. The cryophilic elastase has cracking activity on various gram-positive bacteria, not only can be applied to the fields of medical treatment, daily chemical industry and the like, but also has great application potential in the fields of marine biomedicine, aquatic product processing and the like.

Description

technical field [0001] The invention relates to a deep-sea elastase gene and its preparation method and application, belonging to the technical field of biotechnology. Background technique [0002] Elastase is a proteolytic enzyme characterized by hydrolyzing insoluble elastin, which exists in multiple protease groups, such as serine proteases, cysteine ​​proteases and metalloproteases. Among the metalloproteases, elastase is the most distributed, and elastase exists in M4, M9, M10 and M23 families. At present, elastase is mainly used as a biochemical drug for the treatment of hyperlipidemia and the prevention and treatment of atherosclerosis. The treatment is accurate, safe and reliable. In addition, elastase can also be used in the food industry. Many animal and plant proteins, especially some protein wastes such as ligaments, large arteries and tendons that are difficult to handle and eat, can be degraded by it, so it will be widely used in the deep processing of agricu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/57C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N9/66A01N63/02A01P1/00A61K38/48A61K8/66A61P31/00C12R1/01
Inventor 张玉忠赵慧琳陈秀兰周明扬解彬彬张熙颖周百成
Owner SHANDONG UNIV
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