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Low-nitrogen discharg method for preparing organophosphor hydrolytic enzyme with engineering bacterium fermentation

A technology of engineering bacteria and nitrogen discharge, applied in the direction of hydrolytic enzymes, microbial-based methods, biochemical equipment and methods, etc., can solve problems such as constraints on the development of the fermentation industry, environmental damage, etc., to reduce load and treatment costs, and reduce environmental pollution. The effect of reducing BOD content

Inactive Publication Date: 2011-08-17
浙江凯越生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to obtain a good expression effect, nitrogen excess control is used in the fermentation. The conventional process mostly uses LB medium or a formula derived from it. The nitrogen source is mainly tryptone and yeast powder. Although the production performance is good, but The BOD of the fermentation waste liquid is usually as high as 10,000 or more, causing harm to the environment, and it is also a bottleneck restricting the development of the fermentation industry

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 (shake flask fermentation example)

[0039] The composition and weight ratio of the slant medium are: glucose 0.1%, yeast extract powder 1%, peptone 1%, dipotassium hydrogen phosphate 0.12%, potassium dihydrogen phosphate 0.04%, magnesium chloride 0.1%, sodium chloride 0.3%, agar powder 1.5% -2.0%, the rest is distilled water; adjust the pH to 7.1 with ammonia water; sterilization conditions: 2kg / cm2 (absolute pressure) for 30 minutes; add kanamycin 50u / ml when cooling to below 60°C after sterilization. The inoculum size is 1 inoculation loop connected to 2 standard Petri dishes of 9 cm; the incubation time on the slant is 24 hours.

[0040] First-class strains:

[0041]The medium formula is: peptone 10g, yeast extract powder 3g, ammonium sulfate 10g, glucose 10g, sodium chloride 5g, multivitamin and trace element solution 10mL, distilled water to 1000ml, adjust pH to 6.8; capacity: 1000ml Bottle 200ml of medium, seal with 8 layers of gauze, sterilization...

Embodiment 2

[0051] Embodiment 2 (5 liters fermenter example)

[0052] Cultivate slant strains, the specific method is:

[0053] The composition and weight ratio of the slant medium are: glucose 0.1%, yeast extract powder 1%, peptone 1%, dipotassium hydrogen phosphate 0.12%, potassium dihydrogen phosphate 0.04%, magnesium chloride 0.1%, sodium chloride 0.3%, agar powder 1.5% -2.0%, the rest is distilled water; ammonia water adjusts pH to 7.1; sterilization condition 2kg / cm2 (absolute pressure) steam for 20 minutes; add kanamycin 50u / ml when cooling to below 60°C after sterilization. The inoculum amount is 1 inoculation loop connected to 2 test tube slant surfaces; the slant surface incubation time is 18 hours.

[0054] Cultivate the first-level bacteria, the specific method is:

[0055] The medium formula is: peptone 10g, yeast extract powder 3g, ammonium sulfate 10g, glucose 10g, dipotassium hydrogen phosphate 1.2g, potassium dihydrogenphosphate 0.4g, magnesium chloride 1g, sodium chlor...

Embodiment 3

[0066] Embodiment 3 (150 liters of fermentation tanks feed sugar fermentation)

[0067] Cultivate slant strains, the specific method is:

[0068] Slant medium composition and weight ratio: glucose 0.1%, yeast extract powder 1%, peptone 1%, sodium chloride 0.3%, agar powder 1.5-2.0%, the rest is distilled water; ammonia water adjusts the pH to 7.1; sterilization conditions: 2kg / cm2 (absolute pressure) steam for 25 minutes; add kanamycin 50u / ml when cooling below 60°C after sterilization. The inoculum amount is 1 inoculation loop connected to 3 test tube slant surfaces; the slant surface incubation time is 36 hours.

[0069] First-class strains:

[0070] The specific method of cultivating the first-level strains is:

[0071] The medium formula is: peptone 10g, yeast extract powder 3g, ammonium sulfate 10g, glucose 10g, sodium chloride 5g, multivitamin and trace element solution 1mL, distilled water to 1000ml, adjust pH to 6.8; capacity: 1000ml Bottle 400ml of culture medium...

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PUM

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Abstract

The invention relates to a method for expressing engineering bacteria fermentation, and aims to provide improvement of the method on preparing organophosphorus hydrolase by engineering bacteria fermentation. While the method keeps the level of fermentation, the method has the advantages of less pollution discharge, low load of pollution treatment, simple process and convenient operation. The invention comprises the technical proposal as follows: the method for preparing the organophosphorus hydrolase by engineering bacteria fermentation with low nitrogen emission comprises the following steps: 1) selecting strains; 2) culturing strains in slant; 3) culturing primary strains; 4) culturing secondary strains in a fermentation cylinder; 5) fermenting in the fermentation cylinder; and 6) extracting enzyme powder.

Description

technical field [0001] The invention relates to a method for fermenting and expressing engineering bacteria, in particular to a method for fermenting and preparing organophosphate hydrolase. Background technique [0002] The development of modern society is inseparable from the use of pesticides, but the large-scale use of pesticides has made the problem of pesticide residues increasingly a problem that plagues modern people. To this end, the Institute of Zoology, Chinese Academy of Sciences cultivated a super engineered bacterium [E.coli BL-21(DE3) carrying the plasmid pETDuet-opd-b1], and used the organophosphate hydrolase obtained by inducing the expression of exogenous genes by fermentation, which can effectively degrade Residues of organophosphorus pesticides such as methyl parathion, parathion, methamidophos, and omethoate in fruits and vegetables can also be used as remediation agents for soil and water pesticide pollution, which is one of the achievements of modern b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12R1/19
Inventor 罗天生杨志坚高振岭
Owner 浙江凯越生物科技有限公司