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A marine-sourced collagen-expanding protease vp9 and its coding gene and application

A technology that encodes genes and collagen, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as insufficient fiber unraveling and damage to tanning materials, and achieve good thermal stability, strong salt tolerance, and great application potential Effect

Active Publication Date: 2021-09-24
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the patent still has two major problems, which may cause damage to the tanning substance and insufficient fiber unraveling

Method used

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  • A marine-sourced collagen-expanding protease vp9 and its coding gene and application
  • A marine-sourced collagen-expanding protease vp9 and its coding gene and application
  • A marine-sourced collagen-expanding protease vp9 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Acquisition of Collagen Expansion Protease VP9 Encoding Gene and Construction of Its Expression Strain

[0042] 1. Genomic DNA extraction and whole genome sequencing of Vibrio pomeroyi strain 12613.

[0043] Genomic DNA was extracted from Vibrio pomeroyi strain 12613 according to the instructions of Genome Extraction Kit from Biotec (conventional extraction steps). Whole-genome sequencing was completed by Shanghai Meiji Biotechnology Co., Ltd.

[0044] 2. Purification of extracellular protease secreted by Vibrio pomeroyi strain 12613.

[0045] Vibrio pomeroyi strain 12613 was inoculated into the fermentation medium and cultured at 15°C for 60h. The fermentation broth was centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was dialyzed overnight with 50 mM Tris-HCl buffer (pH 8.0) and then centrifuged at 10000 rpm for 15 min at 4°C. The supernatant was passed through the DEAE-Sepharose Fast Flow chromatography column at a speed of 2.5min / mL and th...

Embodiment 2

[0076] Example 2: Induced Expression and Purification of Collagen Expansion Protease VP9 Heterologous Expression Gene VP9 in Escherichia coli

[0077] 1. Fermentation of recombinant strains

[0078] (1) Cultivation of seeds

[0079] Pick the strain transferred to the recombinant pET-22b-VP9 expression vector on the plate to 100mL LB liquid medium with a final concentration of 100μg / mL ampicillin, and culture overnight at 37°C and 180rpm;

[0080] (2) Inoculate according to the inoculum amount of 1% by volume, and transfer the cultured seed liquid to a shake flask with a liquid volume of 1 L. Cultivate at 37°C and 180rpm until the absorbance of the bacterial solution is 0.8 at a wavelength of 600nm, then change the culture condition to 15°C and 100rpm, continue to cultivate for 30min, then add IPTG (isopropylthiogalactopyranoside) to a final concentration of 0.35mM , continue to cultivate for 16 hours;

[0081] 2. Purification of collagen expansion protease VP9

[0082] (1)...

Embodiment 3

[0086] Embodiment 3: Derived from the property determination of the collagen expansion protease VP9 of wild-type strain and recombinant strain

[0087] 1. Optimum enzyme activity temperature analysis

[0088] The gelatinase activities of the two were measured at 0, 10, 20, 30, 40, 50, 60, 70, and 80°C to determine the optimum reaction temperature of the enzyme.

[0089] The specific method for measuring enzyme activity is as follows: prepare gelatin with a concentration of 2%, take 100 μL of substrate and add 100 μL of diluted enzyme solution, and react at 40° C. for 10 minutes. After the reaction was completed, 200 μL of 1.25 M pre-cooled TCA solution was added to terminate the reaction. Centrifuge at 4°C, 13,000 rpm for 10 minutes, take 20 μL of supernatant, add 100 μL of ninhydrin-sodium citrate mixture, mix well, boil in water bath for 20 minutes, take it out and add 500 μL of 50% n-propanol solution after cooling, mix well and measure at 600 nm color. The control group...

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Abstract

The invention relates to a marine-sourced collagen expansion protease VP9, ​​its coding gene and application. A marine Vibrio strain, Vibrio pomeroyi strain 12613, was deposited in the China Center for Type Culture Collection on March 6, 2020, address: Wuhan, China, Wuhan University, and its strain preservation number is CCTCC NO: 2020050. A coding gene for collagen expansion protease VP9, ​​the nucleotide sequence of which is shown in SEQ ID No.1; the amino acid sequence of collagen expansion protease VP9 expressed by the coding gene is shown in SEQ ID No.2. The collagen-expanding protease VP9 in the present invention has a significant expansion function on collagen, but has no degradative activity on collagen, and has great application potential in the expansion treatment of collagen in leather processing industry and other fields.

Description

technical field [0001] The invention relates to a marine-sourced collagen expansion protease VP9 and its coding gene and application, belonging to the technical field of biotechnology. Background technique [0002] The ocean covers about 71% of the earth's surface, and contains a huge number and variety of marine microorganisms. In order to adapt to the marine environment (i.e., low temperature, high salinity, high pressure), marine bacteria have evolved a variety of physiological mechanisms and produced a large number of enzymes with unique properties and functions. Therefore, marine microorganisms have become a huge treasure trove for the development of new industrial and medical enzymes. Screening and isolating proteases with special mechanism of action and unique functions from marine microorganisms has become one of the research hotspots. Discovering new proteases in marine microorganisms and studying their functions is of great significance to the development and uti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/52C12N15/57C12N15/70C12N1/21C07K14/78C12R1/63
CPCC07K14/78C12N9/52C12N15/70C12N1/205C12R2001/63
Inventor 张玉忠王琰陈秀兰刘白雪宋晓妍
Owner SHANDONG UNIV
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