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Collagenase producing strain as well as collagenase gene sequence and application thereof

A technology of collagenase and bacterial strains, which is applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of difficult to fully utilize, difficult digestion and absorption of collagen macromolecules, and achieve a wide range of pH applications, strong temperature tolerance, The effect of high enzyme activity

Inactive Publication Date: 2017-07-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its unique three-strand superhelical structure, its properties are very stable, and it cannot be decomposed by general processing temperature and short-term heating, and its high molecular weight makes it difficult to digest and absorb collagen macromolecules, and is not easy to be fully utilized by the human body.

Method used

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  • Collagenase producing strain as well as collagenase gene sequence and application thereof
  • Collagenase producing strain as well as collagenase gene sequence and application thereof
  • Collagenase producing strain as well as collagenase gene sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 strain Col15 Screening, identification and enzyme-producing properties of

[0029] 1. Screening of collagenase-producing strain Col15

[0030] The applicant used soil samples collected near the Tianjin Meat Factory as the matrix, mixed them with sterile water and egg whites in a certain proportion, then added small pieces of pig skin and fish skin, and enriched at 25-30 °C Cultured; then diluted and spread to contain 2 % gelatin The LB flat plate was re-screened by the transparent circle of acidic mercury reagent, and then the strains of the primary screening were passed through the shake flask, and the enzyme activity was measured respectively, and finally the bacterial strain of the present invention was obtained.

[0031]2. Identification of collagenase-producing strain Col15

[0032] 1. Morphological characteristics of collagenase-producing strain Col15

[0033] The colony of the collagenase-producing strain Col15 in the present invention is w...

Embodiment 2

[0047] Example 2 strain Col15 Fermentation optimization research

[0048] The fermentation medium and fermentation conditions for the production of collagenase by strain Col15 were optimized by single factor experiments. Add 50 mL of fermentation medium to a 250 mL Erlenmeyer flask, sterilize with high pressure steam at 121 °C for 30 min, and sterilize the monosaccharides in the medium separately, and then add them to the medium after sterilization and cooling. All experiments were designed in triplicate, and the optimization results of the previous step were used in subsequent experiments.

[0049] (1) Determination of the optimum carbon source for the medium

[0050] When preparing the culture medium, 2% of different carbon sources were added, and other components remained unchanged. The enzyme activity was determined after 54 hours of fermentation in shake flasks. The results showed that the optimum carbon source of Col15 strain was glucose, and other carbon sour...

Embodiment 3

[0059] Example 3 strain Col15 Fish skin degradation experiment

[0060] After the strain Col15 was revived and activated, it was fermented at 37 °C and 200 rpm for 54 h, centrifuged at 5500 rpm for 30 min, and the supernatant was collected to obtain the crude enzyme solution of the strain Col15. The culture medium of the uncultivated strain Col15 was used as a control.

[0061] Thaw cod skin with tap water, remove excess minced meat and fish scales, cut into 5 mm x 5 mm pieces, fully drain, weigh and divide into two equal parts, add the drained equal parts of fish skin to In the Erlenmeyer flask containing 50 mL of crude enzyme solution and control medium, react in a water bath shaker at 37 °C and 120 rpm for 48 h. With reference to the control, it can be clearly seen that the cod skin has degraded.

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Abstract

The invention relates to a collagenase producing strain, namely bacillus cereus, heterologous expression of a collagenase gene thereof and application thereof. The strain is separated and sieved from a soil sample and belongs to bacillus cereus after being identified. The strain is capable of extracellularly producing protease with collagen hydrolytic activity, and the protease with a molecular weight of 100kDa and collagenase activity is obtained by separating and purifying thallus fermentation supernate. Meanwhile, the collagenase gene of the strain is obtained through gene mining, and a collagenase protein with a molecular weight of 100kDa is obtained through heterologous expression and purification of escherichia coli. Researches confirm that the collagenase obtained through fermentation and purification of the Col15 strain and the collagenase obtained through heterologous expression and purification of escherichia coli are the same protein containing signal peptide. The collagenase produced by bacillus cereus Col15 has the properties of relatively wide temperature tolerance and pH value tolerance range and the like. Products obtained by hydrolyzing collagen and analogues thereof by virtue of the strain or the collagenase have wide economic prospects in the industries of foods, cosmetics, medical treatment and the like.

Description

technical field [0001] The invention belongs to the technical field of industrial microorganism screening application, in particular to a strain producing collagenase, and at the same time relates to a collagenase coding gene colc and its amino acid sequence, as well as the application of its encoded collagenase in the process of collagen degradation. Background technique [0002] Collagen, also known as collagen, English name Collagen, exists as a structural protein in the extracellular matrix. It is a white, opaque, unbranched fibrous protein with a molecular weight of about 300 kDa. It is widely present in animal bones, feet, cartilage, skin and other connective tissues in the form of insoluble fibers, accounting for about one-third of the total protein content of mammals, equivalent to 6% of body weight. In animals, its main function is to maintain the shape and structure of skin and tissues and organs, to support organs and protect the body. Therefore, collagen is wide...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/54C12N15/57C12N15/70C12N15/75C12P21/06A61K38/39A61P1/14A61P9/12A61P39/06A61P19/10A61K8/65A61Q19/08A23L33/18C12R1/085
CPCA61K8/65A61Q19/08C12N9/54C12P21/06C12Y304/24003
Inventor 朱玥明李星硕曾艳门燕管于平柏玮孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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