Recombinant engineering strain of anammox bacterium diamine oxidase

A technology of anaerobic ammonium oxidizing bacteria and recombinant engineering bacteria, applied in the direction of oxidoreductase, genetic engineering, recombinant DNA technology, etc., can solve problems such as harsh growth conditions, polluted water bodies, affecting human activities and health, and achieve increased expression and functional activity, and the effect of shortening the expression time

Inactive Publication Date: 2015-04-08
SHANGHAI UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the natural anammox bacteria grow slowly, the period of time is as long as 11 days, and the growth conditions are harsh, so they cannot be enriched and cultivated at a high concentration in a short time, so that hydrazine can be oxidized into the final environmentally friendly product N 2 The application of the anammox bacteria hydrazine oxidase HZO in the anammox reaction process is limited, resulting in the accumulation of a large amount of hydrazine (also known as hydrazine) in the bacteria. Because hydrazine is extremely toxic, when it is released from the bacteria into the water body It will seriously pollute the water body, thus affecting human activities and health

Method used

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  • Recombinant engineering strain of anammox bacterium diamine oxidase
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  • Recombinant engineering strain of anammox bacterium diamine oxidase

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Experimental program
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Effect test

Embodiment 1

[0026] The hydrazine oxidase HZO gene sequence was amplified, and the primers were as follows:

[0027] Forward primer hzoF1:5 ， -TGTGCATGGTCAATTGAAAG-3 ， ;

[0028] reverse primer hzoR1:5 ， -CAACCTCTTCWGCAGGTGCATG-3 ， .

[0029] 5 of upstream and downstream primers ， A restriction site was designed at the end for connection with the expression vector pGEM-T. The gene sequence of hydrazine oxidase HZO was amplified by PCR with primers containing restriction sites. The specific system and amplification conditions are as follows:

[0030] PCR reaction system for amplifying HZO:

[0031]

[0032] PCR amplification conditions (or colony PCR amplification conditions) are:

[0033] Pre-denaturation at 94°C for 5 minutes

[0034]

[0035] Construction of pGEM-T-HZO recombinant vector:

[0036] The PCR purified product containing the ammonia oxidase HZO gene sequence was connected to the pGEM-T vector by DNA ligase overnight at 4°C, and the ligated product was introdu...

Embodiment 2

[0040] Preservation of Gene Recombined Engineering Bacteria

[0041] Take a clean 250mL Erlenmeyer flask, add ultrapure water:glycerol=50:50 ultrapure water and glycerin into it, wait until the solution is evenly mixed, and sterilize at 120°C for 20 min. Add 1800 μL and 1200 μL of the sterilized glycerin aqueous solution and recombinant engineered bacteria to a 5mL sterilized centrifuge tube (to ensure that the glycerol content in the final sterile glycerol aqueous solution is 30%), mix the solutions evenly, and store them at -80°C.

Embodiment 3

[0043] Application of Gene Recombination Engineering Bacteria

[0044] Use LB liquid medium to expand (20 times) to cultivate HZO gene recombinant engineered bacteria to OD 600 reached 0.6. Take 20 mL of bacterial liquid, apply it to 100 mL of hydrazine-contaminated solution with an initial concentration of 45 mg / L, and shake at 200 rpm for 28 hours at 37°C. Take 5mL of the reaction solution and pass it through a 0.22μm filter membrane, and use p-dimethylaminophenol spectrophotometry to detect the concentration of residual hydrazine in the solution during the reaction, and the calculated removal rate is 98%. At the same time, take 5mL of the reaction solution and detect it with a UV-4802 spectrophotometer, using OD 600 The value represents the growth of the bacterium, and the growth of the HZO gene recombination engineered bacteria showed a complete S-shaped growth curve after testing.

[0045] Shanghai University

[0046] Recombinant engineering bacteria of anammox bacter...

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Abstract

The invention relates to a recombinant engineering strain of anammox bacterium diamine oxidase. The recombinant engineering strain has a base sequence represented by SEQ ID No. 1. According to the engineering strain disclosed by the invention, the expression time of the anammox bacterium diamine oxidase is shortened, the expression level and functional activity of the gene are greatly improved, and the application of the anammox bacterium diamine oxidase to an anaerobic ammonia oxidation process is realized. The recombinant engineering strain of the anammox bacterium diamine oxidase is fermented and then is applied to the treatment of wastewater with a low carbon-nitrogen ratio.

Description

technical field [0001] The invention relates to a recombinant engineering bacterium of anammox hydrazine oxidase and a preparation method thereof. Background technique [0002] With the development of social economy, due to the provision of living standards and the improvement of diet structure, the carbon-nitrogen ratio of domestic sewage is unbalanced. For domestic sewage with low carbon-to-nitrogen ratio, most of the secondary sewage treatment plants that operate according to the original design water quality cannot meet the discharge standards, which causes a large waste of resources and funds for sewage plants. At the same time, when the traditional nitrification / denitrification process treats sewage with a low carbon-to-nitrogen ratio, due to insufficient carbon sources, the nitrification / denitrification process in the biological denitrification process is incomplete, resulting in a large amount of nitrite accumulation and low denitrification efficiency. The total nit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12N15/53
CPCC12N9/0044C12N15/70C12Y107/99008
Inventor 陆永生陈学萍杨兴兴刘冬秀钱光人
Owner SHANGHAI UNIV
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