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Thermophilic neutral protease gene, engineering bacteria, enzyme and application thereof

A technology of neutral protease and gene, applied in the field of thermophilic neutral protease gene, engineering bacteria, enzyme and its application, can solve problems such as complex conditions, limited application range, poor stability, etc.

Inactive Publication Date: 2017-09-15
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Enzyme has complex conditions in practical application, and the disadvantage of neutral protease is its poor stability, which limits its application range

Method used

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  • Thermophilic neutral protease gene, engineering bacteria, enzyme and application thereof
  • Thermophilic neutral protease gene, engineering bacteria, enzyme and application thereof
  • Thermophilic neutral protease gene, engineering bacteria, enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: Construction and protein expression of thermophilic neutral protease engineering bacteria, comprising the following steps:

[0060] (1) Cultivation of thermophilic bacteria Thermus thermophilus HB8 and extraction of genomic DNA

[0061] The composition of 1L thermophilic bacteria culture medium is: peptone 4.0g, yeast extract 2.0g, NaCl 1.0g, Castenholz salt solution 10.0mL, distilled water 1.0L, adjust the pH value to 7.2, 1.034×10 5 Pa high pressure steam sterilization for 20min.

[0062] Castenholz salt solution: nitrilotriacetic acid 1.0g, CaSO 4 2H 2 O 0.6g, MgSO 4 ·7H 2 O 1.0g, NaCl 0.08g, KNO 3 1.03g, NaNO 3 6.89g, Na 2 HPO 4 1.11g, FeCl 3 ·6H 2 O solution (mass fraction 0.03%) 10.0mL, Nitsch's trace elements 10.0mL, distilled water 1.0L, adjust the pH to 8.2, and filter to sterilize.

[0063] Nitsch's Trace Elements: H 2 SO 4 0.5mL, MnSO 4 ·xH 2 O 2.2g, ZnSO 4 ·7H 2 O 0.5g, H 3 BO 3 0.5g, CuSO 4 ·5H 2 O 0.016g, Na 2 MoO 4...

Embodiment 2

[0092] Example 2: Properties of Recombinant Thermoneutral Protease and Mature Thermoneutral Protease

[0093] (1) Optimum reaction temperature and temperature stability

[0094] Temperature is an important factor affecting the catalytic activity of enzymes. Using casein as a substrate, the activity of the recombinant thermophilic neutral protease of the present invention is measured within the range of 50-90°C, and the relative activity of the enzyme is plotted against the temperature, such as Figure 4 It is shown that the optimum reaction temperature of the thermophilic neutral protease is 85°C, and more than 80% of the enzyme activity can be maintained in the range of 65-90°C. The activity-temperature curve of the mature thermophilic neutralase is shown in Figure 5 , its optimum reaction temperature is 75°C, and in the range of 65-85°C, the enzyme activity is maintained at a relatively high level, close to the highest activity; in the range of 50-90°C, the mature thermop...

Embodiment 3

[0104] Example 3: Analysis of Thermophilic Neutral Protease Degraded Proteins

[0105] In order to detect the degradation ability of thermophilic protease on different protein substrates, 50 μL of 0.1 mg / mL thermophilic protease was mixed with 5 mg / mL casein and 2.5 mg / mL BSA respectively and incubated at 85 °C After 15min, the substrate degradation was observed by SDS-PAGE ( Figure 12 ). Electrophoresis showed that casein and BSA were all degraded within 15 minutes, indicating that the thermophilic neutral protease has strong proteolytic ability and has high application potential in the application field of protein degradation.

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Abstract

The invention discloses a thermophilic neutral protease gene, engineering bacteria JD-TP (Escherichia coli) producing thermophilic neutral protease, thermophilic neutral protease produced by fermenting the engineering bacteria, mature thermophilic neutral protease after self-catalytic shearing and an application of the thermophilic neutral protease and the mature thermophilic neutral protease in degrading proteins, and belongs to the technical field of bioengineering. The thermophilic neutral protease gene is obtained by amplifying a thermophile Thermus thermophilus HB8 genome and is connected to an expression vector to be successfully expressed in escherichia coli; the optimum reaction temperature of the thermophilic neutral protease is 85 DEG C, the half life at 85 DEG C is 8d, and the optimum reaction temperature of the mature thermophilic neutral protease is 75 DEG C; the optimum pH of the thermophilic neutral protease is 7.5-10.0, and the optimum reaction pH of the mature thermophilic neutral protease is 6.5; the thermophilic neutral protease and the mature thermophilic neutral protease have very high optimum reaction temperatures, thermal stability and relatively wide reaction pH values and have a very good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a thermophilic neutral protease gene, an engineering bacterium JD-TP (Escherichia coli) producing the thermophilic neutral protease, and a gene produced by fermentation of the genetically engineered bacterium. A thermoneutral protease, a mature thermoneutral protease obtained after self-catalyzed cleavage, a use of the thermoneutral protease and the mature thermoneutral protease in degrading proteins. Background technique [0002] Protease is a kind of hydrolytic enzyme that breaks the peptide bond in macromolecular protein to make it into small molecular protein or polypeptide. According to the type of amino acid residues in the active center, it can be divided into serine protease, cysteine ​​protease, aspartic acid protease and metalloprotease. According to the pH value of the reaction, it can be divided into acid protease, neutral protease and alkaline pro...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N1/21C12N15/70C12P21/06C12R1/19
CPCC12N9/52C12N15/70C12P21/06
Inventor 解桂秋高仁钧李杏杏宗利霍锐陈曼利
Owner JILIN UNIV
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