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Novel low-temperature protease coding gene and functional expression of novel low-temperature protease coding gene in Escherichia coli

A low-temperature protease and encoding gene technology, applied in the field of novel low-temperature protease encoding gene and its functional expression in Escherichia coli, can solve the problems of low optimal enzyme activity temperature, rare low-temperature protease, poor thermal stability of low-temperature protease, etc.

Active Publication Date: 2015-07-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(2) Low-temperature protease has a low optimum enzyme activity temperature. Currently, the optimum catalytic temperature of the protease isolated from low-temperature bacteria is 25°C, but most of the optimum catalytic temperatures are between 30°C and 40°C. , generally 10 to 20°C lower than the optimum enzyme activity temperature of mesophilic protease
(3) Low-temperature proteases have poor thermal stability. It has been found that almost all low-temperature proteases are sensitive to heat, which is mainly due to the structural characteristics of enzyme molecules.
However, there are still few examples of obtaining low-temperature proteases by this method

Method used

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  • Novel low-temperature protease coding gene and functional expression of novel low-temperature protease coding gene in Escherichia coli
  • Novel low-temperature protease coding gene and functional expression of novel low-temperature protease coding gene in Escherichia coli
  • Novel low-temperature protease coding gene and functional expression of novel low-temperature protease coding gene in Escherichia coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Cloning of the whole cpls8 gene

[0046] According to the strain classification identification and protein sequencing results, degenerate primers were designed:

[0047] cpls8F1: 5'-CGAATTRTWGGTGGGAAAAACT-3'

[0048] cpls8R1: 5'-TGYGGYGTYGCCATTGAAGT-3'

[0049] The marine bacterium Planococcus sp.11815 genome was extracted, and a 565bp conserved region in the cpls8 gene was amplified by PCR using it as a template. The PCR amplification conditions were: 95°C for 5 minutes, 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, 30 cycles, and 72°C for 5 minutes. According to the sequencing and analysis results of the conserved region, redesign the reverse primer:

[0050] cpls8F2: 5'-GATTATAACGGGCATGGCACC-3'

[0051] cpls8R2: 5'-CCGGACAGACTGGCATATTTC-3

[0052] Partial digestion of Planococcus sp.11815 genomic DNA with Mbo I, electrophoresis separation and recovery of 2-3 kb fragments, ligation and circularization at 16°C, and using this as a template to amplif...

Embodiment 2

[0054] Sequence Analysis of the Whole Gene of cpls8

[0055] The Blast and ORF Finder tools on the NCBI website were used for DNA sequence analysis, alignment and open reading frame positioning, and for protein classification and functional analysis. http: / / merops.sanger.ac.uk / , promoter and expression regulatory sequence prediction using http: / / www.cbs.dtu.dk / services / Promoter / Promoter 2.0Prediction Server tool.

Embodiment 3

[0057] Expression of cpls8 Gene in Escherichia coli E.coli Rosetta(DE3)

[0058] According to the sequence of the ORF region of the cloned gene cpls8, the upstream and downstream primers cpls8F-Nde I and cpls8R-Xho I were designed respectively, and Nde I and Xho I restriction sites were added at the end of the primers.

[0059] cpls8F-Nde I:

[0060] 5'-GGGAA TTCCAT ATGAAAAATATTCATTTGATTCCATACCGC-3'

[0061] cpls8R-Xho I:

[0062] 5'-CCG CTCGAG CCGGTCCAATCCATTAGCATAAGA-3'

[0063] Using the genome of Planococcus sp.11815 as a template, the open reading frame (ORF) of cpls8 was amplified by PCR (PCR conditions: 95°C for 5 minutes, 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, 30 cycles, 72°C extension for 5 minutes), recovered the PCR product, and cloned it into the expression vector pET22b using the Nde I and Xho I restriction sites to obtain the expression vector pET22b-cpls8, the amino acid sequence expressed by the gene Form a fusion protein with a hi...

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Abstract

The invention relates to a novel low-temperature protease coding gene and functional expression of the novel low-temperature protease coding gene in Escherichia coli. According to the invention, a novel low-temperature protease coding gene segment cpls8 is cloned from planococcus sp.11815, and sequence analysis shows that a protein sequence coded by the novel low-temperature protease coding gene contains 329 amino acid residues in total, belongs to S8 family serine protease, and maximum pramariness with a published protein sequence in an existing database is 77% only. The novel low-temperature protease coding gene realizes functional expression in E.coil Rosseta (DE3), and enzymatic property analysis shows that optimal catalysis pH value is 10, optimal catalysis temperature is about 37 DEG C, and more than or equal to 50% of maximum enzyme activity can be maintained at the temperature of 20-30 DEG C, so that the novel low-temperature protease coding gene belongs to typical low-temperature alkaline protease and has a broad application prospect in the fields of washing, foods, medicines and the like.

Description

technical field [0001] The invention belongs to the field of gene cloning and expression, in particular to a novel low-temperature protease coding gene and its functional expression in Escherichia coli. Background technique [0002] Protease is a general term for a large class of enzymes that hydrolyze proteins. It exists widely in animals, plants and various microorganisms. Since the last century, it has been widely used in food, washing, leather, feed, paper, pharmaceutical and other industries, accounting for More than 60% share of the world enzyme market. The proteases currently used are generally medium-high temperature proteases, and the optimum enzyme activity temperature is generally around 50°C, and the optimum growth and enzyme production temperature of enzyme-producing bacteria is above 30°C. Low-temperature protease refers to a class of proteolytic enzymes whose optimum enzyme activity temperature is below 40°C and which can still maintain high catalytic activit...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/52C12N1/21C12R1/19
Inventor 路福平张会图莫清珊
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY