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Cryophilous proteinase gene mcp01 and its prepn process

A technology for cold-adapting protease and protease, which is applied in the field of new deep-sea cold-adapting protease gene and its preparation, and can solve the problems of difficulty in sampling and cultivation, and few researches on deep-sea microorganisms, etc.

Inactive Publication Date: 2007-10-03
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the difficulty of sampling and culturing, there are relatively few studies on deep-sea microorganisms.

Method used

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  • Cryophilous proteinase gene mcp01 and its prepn process
  • Cryophilous proteinase gene mcp01 and its prepn process
  • Cryophilous proteinase gene mcp01 and its prepn process

Examples

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Effect test

Embodiment 1

[0192] The coding gene fragment sequence of the novel deep-sea cryogenic protease deseasin MCP-01 is SEQ ID NO.1 as described above. The corresponding relationship between nucleotides and encoded amino acids in genes is also as described above. The genome DNA fragment has a total of 2676bp, which contains a 2508bp open reading frame encoding protease deseasin MCP-01, the start codon is located at 143bp, the stop codon is located at 2648bp, and a total of 835 amino acids are encoded.

Embodiment 2

[0193] Embodiment 2: gene fragment cloning method of the present invention

[0194] Source of strain: Pseudomonas sp.SM9913 was isolated from seabed sediments at a depth of 1855 meters. See Chen Xiulan et al., "Low-temperature protease produced by deep-sea psychrotroph SM9913", "Marine Science" 2001, Vol. 5, No. 1, pp. 4-8.

[0195] The specific method is as follows:

[0196] 1. Purification of MCP-01

[0197] (1) The Pseudomonas sp.SM9913 bacterial suspension was inoculated in the fermentation medium (corn flour 2%, bran 1%, soybean meal 2%, Na 2 HPO 4 0.4%, K 2 PO 4 0.03%, CaCl 2 0.1%, aged seawater (pH7.5), 50ml / 500ml, 12°C, 180 rpm, cultured for 72 hours.

[0198] (2) After culturing, collect the culture medium, centrifuge at 11000 rpm for 15 minutes at 4°C, take the supernatant, add ammonium sulfate powder to 55% saturation while stirring in an ice bath, then 10,000 rpm at 4°C Centrifuge for 1 minute, remove the supernatant, and dissolve the pellet in 50 mM Tri...

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Abstract

The present invention is one kind of cryophilous protease gene mcp01 and its preparation process, and belongs to the field of biotechnology. The cryophilous proteinase gene mcp01 has one genome DNA segment of 2676 bp containing one opened reading frame with 2508 nucleotides. The opened reading frame is the gene encoding protease MCP-01, and the protease MCP-01 gene mcp01 expressed cryophilous protease has optimal enzyme activity temperature of about 35 deg.c and optimal enzyme pH of 9.0. The present invention may be applied in low temperature preservation of meat, dairy product processing, detergent production, etc.

Description

technical field [0001] The invention relates to a gene of a novel deep-sea cold-adapted protease and a preparation method thereof, belonging to the technical field of marine biology. Background technique [0002] Proteases, proteinases, also known as peptidases, are a class of hydrolytic enzymes that catalyze the breaking of peptide bonds. Protease widely exists in animal viscera, plant stems, leaves and fruits. A variety of microorganisms can secrete extracellular protease, which is the main source of protease in industrial production. According to different modes of action, proteases are divided into exopeptidases and endopeptidases. Exopeptidases cleave one or several amino acids at the N- or C-terminus of a polypeptide chain. Therefore, there are two types of exopeptidases: aminopeptidases (cleaved from the N-terminus) and carboxypeptidases (cleaved from the C-terminus). Endopeptidases cleave peptide bonds inside polypeptide chains. According to different catalytic m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/52
Inventor 陈秀兰张玉忠张熙颖何海伦石梅
Owner SHANDONG UNIV
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