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A strain for producing medium and low temperature β-mannanase and its application

A technology of mannanase and low temperature, which is applied in the field of microorganisms, can solve the problems of cost limitation on the application scale of β-mannanase, and achieve the effect of meeting the needs of oil field fracturing engineering, wide temperature tolerance range and easy availability of raw materials

Active Publication Date: 2015-11-11
大连知微生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of pH adaptability, alkaline β-mannanase has also been patented, but it is currently only used in detergents and food and feed industries (Ma Yanhe et al. publication: CN1266096)
[0012] In addition, low production and high cost limit the scale of application of β-mannanase in oil and gas reservoir development. One of the most important ways to change this situation is to screen and breed highly active and alkali-resistant β-mannanase. - Mannanase high-producing enzyme strains

Method used

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  • A strain for producing medium and low temperature β-mannanase and its application
  • A strain for producing medium and low temperature β-mannanase and its application
  • A strain for producing medium and low temperature β-mannanase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Isolation and Identification of Example 1 Strain OPUS-001

[0050] 1. Isolation of strain OPUS-001

[0051] (1) Sample collection: from the root soil of plants on the banks of Hongjiannao Lake in Shenmu County, Shaanxi Province;

[0052] (2) Heat treatment: the soil sample was made into a suspension with sterile deionized water, and heat treated at 80°C for 10 minutes to kill the bacteria;

[0053] (3) Enrichment culture: add nutrient sources to the heat-treated suspension, and culture at 37°C for 2 days to obtain enriched spore-forming bacteria;

[0054] (4) Separation and purification: gradiently dilute the enriched cultured microbial culture solution, and select a dilution factor of 10 -5 、10 -6 and 10 -7 The three gradients were streaked on solid medium and incubated overnight at 37°C. Pick a single colony and transfer it to the seed medium, and culture it with shaking at 37°C for 1 day to become the seed solution;

[0055] (5) Introduce the seed liquid into t...

Embodiment 2

[0061] The fermentation of embodiment 2OPUS-001 strain

[0062] Inoculate the OPUS-001 strain on the guar gum solid medium and culture it at 37°C for 2 days; inoculate a single colony of the solid medium into the seed medium with a volume ratio of 1 / 5-1 / 4, and incubate at 37°C Cultivate with shaking at 150 rpm for 1 day at °C to become a seed liquid; add the seed liquid to the fermentation medium at a volume ratio of 1 / 50, and culture at 37 °C for 3 days to obtain a fermentation liquid.

Embodiment 3

[0063] The separation and purification of embodiment 3β-mannanase

[0064] Use the following method to separate and purify the β-mannanase in the fermentation broth of OPUS-001 strain:

[0065] (1) Centrifuge the fermentation broth of Example 1 at 3000 rpm and 4°C for 15 minutes, take the supernatant, and filter to obtain a crude extract;

[0066] (2) Add 10 times the amount of 45-65% saturated ammonium sulfate to the crude extract, put the solution on a magnetic stirrer at 4°C overnight; centrifuge at 20,000rpm and 4°C for 30min, keep the supernatant; Add saturated ammonium sulfate to 0.5:1 (v / v), centrifuge again to obtain a precipitate; dissolve the precipitate in PBS, dialyze with PBS solution to remove ammonium sulfate, and obtain a dialysis enzyme solution;

[0067] (3) The dialysis enzyme solution in step (2) was separated by Sepharose G-75 column chromatography, and after loading the sample was eluted with 0.1M NaCl aqueous solution, and the eluate was collected, each...

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Abstract

The invention discloses bacillus subtillis OPUS-001 for producing medium-low-temperature beta-mannase. Preservation number of the bacterial strain in China General Microbiological Culture Collection Center is CGMCC No.7361. The bacterial strain disclosed by the invention is simple in culture method, raw materials are easily available, enzyme yield of the bacterial strain is strong; produced alkali beta-mannase enzyme preparation is high in activity, alkaline-resistant and salt-resistant, has efficient gel breaking capability for oil field guar gum or modified guar gum fracturing fluid under a medium-low region temperature condition, is free of incompatibility with most of chemical assistants in the fracturing fluid, and can satisfy needs of oil field fracturing projects.

Description

technical field [0001] The invention belongs to the technical field of microbes, and relates to a strain producing β-mannanase and its fermentation, enzyme separation and purification, application of the strain in medium and low temperature well hydraulic fracturing and gel breaking, and preparation of β-mannan by using the strain Glycanase method. The β-mannanase has the characteristics of mesophilic hypothermia and alkali resistance. Background technique [0002] Hydraulic fracturing is an important technical measure for increasing production of oil and gas wells and increasing injection of water wells. When the surface high-pressure pump unit injects high-viscosity liquid into the well at a displacement that greatly exceeds the absorption capacity of the formation, and the pressure near the bottom of the well exceeds the in-situ stress near the well wall and the tensile strength of the rock, a fracture is formed in the formation. As the proppant-laden fluid is injected ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/42C09K8/60C12R1/125
Inventor 赵静
Owner 大连知微生物科技有限公司
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