Medium and low temperature endo-β-mannanase and its coding gene and application

A technology of mannanase and low temperature, applied in the field of genetic engineering, can solve the problems of unfriendly environment, generation of pollutants, unsuitable application of chemical gel breaker engineering, etc., and achieve remarkable economic benefits

Active Publication Date: 2022-01-18
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since this reaction has requirements on temperature and time, the minimum temperature of the reaction is 50°C to achieve gel breaking within 1 hour, which is only applicable to medium-high temperature reservoirs, and for medium-low temperature reservoirs with a temperature lower than 50°C , chemical breakers are not suitable for engineering applications
In addition, the oxidation reaction required for chemical gel breaking is random, and it will react with pipelines, foundation materials, hydrocarbons, etc. to produce pollutants, which is not friendly to the environment and has many limitations [Zhuang Zhaofeng, Zhang Shicheng, Zhang Jin, et al. Research and application of medium-high temperature and low-concentration fracturing fluid[J].Petrochemical,2007,24(2):120-123]

Method used

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  • Medium and low temperature endo-β-mannanase and its coding gene and application
  • Medium and low temperature endo-β-mannanase and its coding gene and application
  • Medium and low temperature endo-β-mannanase and its coding gene and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Gene Cloning of Low Temperature Endo-β-Mannanase ManBL27-1 and ManBL27-2 in Example 1

[0040] Genomic DNA of Bacillus subtilis BL-27 was extracted according to the operation steps of the bacterial genomic DNA extraction kit (spin column type Cat#: DP2001). After comparing the complete gene sequence of Bacillus subtilis BL-27 with the β-mannanase gene sequence in NCBI database, primers BL27man1-F:5'-CATGCCATGGCCTTTAAGAAACATACGATCTCTT-3' and BL27man1-R:5 were designed '-CCGCTCGAGTTCAACGATTGGCGTTAAAGAA-3' was used to amplify the complete coding gene with signal peptide; primers BL27man2-F: 5'-CATGCCATGGCCACTGTGTCGCCTGTGAATCCTA-3' and BL27man2-R: 5'-CCGCTCGAGTTCAACGATTGGCGTTAAGAA-3' were designed to amplify and remove the signal The gene encoding the peptide. Using the extracted genomic DNA of Bacillus subtilis BL-27 as a template, amplify the gene sequence encoding β-mannanase BL27Man, the gene manBL27-1 containing signal peptide and the gene manBL27-2 without signal pep...

Embodiment 2

[0041] Recombinant expression of embodiment 2 gene manBL27-1 and manBL27-2 in Escherichia coli BL21 (DE3)

[0042] Taking pET28a as the expression vector, NcoI and XhoI restriction sites were added to the designed upstream and downstream primers respectively as the action sites for constructing the recombinant expression vector. The genes manBL27-1, manBL27-2 and expression vector pET28a recovered from the PCR gel were double-enzyme-digested with NcoI and XhoI respectively (50 μL double-digestion system: 30 μL fragment or plasmid, 2 μL NcoI fast-cut enzyme, 2 μL XhoI fast-cut enzyme, 10 μL Quick-cut Enzyme Buffer, 6 μL ddH 2 O), after enzyme digestion product gel recovery, use T 4 DNA ligase ligation (25 μL ligation system: T4 DNA Ligase 1 μL, 10×T4 DNA Ligase Buffer 2.5 μL, DNA fragment about 0.3 pmol, carrier DNA about 0.03 pmol, ddH 2 O up to 25μL), ligated overnight at 16°C. Take 10 μL of the ligation product to transform 50 μL of E.coli BL21(DE3) competent cells, spre...

Embodiment 3

[0044] Example 3 Determination of Enzyme Activity of Recombinantly Expressed β-Mannanase (DNS Method)

[0045] Preparation of mannose standard curve: Take a clean graduated test tube and label it, draw 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000μL of 1g / L standard mannose solution in a 10mL test tube respectively, The one without mannose was used as a blank control, and three parallels were done for each sample. Add distilled water to each test tube to make up to 1mL, then add 3mL DNS reagent each, boil for 4min, cool with running water, then distill the volume to 15mL with distilled water, and measure the absorbance at a wavelength of 540nm. According to the data to make a standard curve, such as Figure 5 shown.

[0046] Determination of enzyme activity: select locust bean gum, konjac gum, squash gum, guar gum, hydroxypropyl guar gum, and cationic guar gum as substrates, and first use 50mM pH7 phosphate buffer to prepare 8g / L glue solution. Add 100 μL of crude enz...

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Abstract

The present invention relates to a medium-low temperature endo-β-mannanase and its coding gene and application, the nucleotide sequence of which is shown in SEQ ID NO: 1 or SEQ ID NO: 2. The β-mannanase of the present invention is derived from Bacillus subtilis BL-27, and the β-mannanase expressed by the recombinant bacteria has a very low enzymatic activity, which can quickly reduce the viscosity of guar gum but only produce a very small amount of Mannan oligosaccharides have huge differences in enzymatic properties from existing β-mannanases; β-mannanase ManBL27-1 can convert water at 40-50°C within 1 hour without breaking cells The viscosity of the base fracturing fluid drops from above 5000mpa.s to 500mpa.s, and the viscosity can be completely reduced with the increase of time. The β-mannanase ManBL27-2 genetically engineered bacteria has formed a "self-embedding" of the bacteria, Under the same conditions, the viscosity-reducing rate of water-based fracturing fluid has an obvious tendency to slow down, and the slow-release viscosity-reducing effect of mannanase in water-based fracturing fluid system is realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular, the invention relates to an endo-β-mannanase with medium and low temperature activity and alkali resistance and its gene and the use of the β-mannanase as a water-based fracturing fluid Application of breakers. Background technique [0002] With the exploration and development of oil in our country, the reserves of low-permeability oil reservoirs account for more than two-thirds of the total oil reserves, and it is increasing year by year; and most of the oil consumption now comes from low-permeability oil reservoirs , since then, the development and utilization of low-permeability reservoirs is an important goal in today's oil production. The hydraulic fracturing technology, which was born in 1949, has become a general technology for the exploitation of low-permeability reservoirs, which can effectively stimulate low-permeability oil wells and restore their oil produc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/70C09K8/68C09K8/90
CPCC12N9/2434C12Y302/01025C12N15/70C09K8/905C09K8/68C09K2208/24
Inventor 李霜吕亮陶惟一王丹
Owner NANJING TECH UNIV
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