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Pseudomonas sp. as well as preparation method and application of dual-functional enzyme preparation of pseudomonas sp.

A Pseudomonas, bifunctional enzyme technology, applied in the field of environmental biology, to achieve a significant effect of degradation activity

Active Publication Date: 2017-10-24
ECOLOGY INST SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, multifunctional enzymes that can efficiently degrade different types of POPs at the same time are still blank at home and abroad.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A strain of Pseudomonas sp. ECO-1 was deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on March 31, 2017, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, deposit number: CGMCC No.13960.

[0033] The above-mentioned Pseudomonas sp. (Pseudomonas sp.) ECO-1 strain was screened and obtained by the following method:

[0034] Take the leachate from POPs-contaminated soil and dilute it to a concentration of 10 -1 、10 -2 、10 -3 、10 -4 、10 -55 concentration gradients. Spread the diluted bacterial suspension on the solid medium containing polychlorinated biphenyls and atrazine, do two parallels for each concentration, and culture at 30°C for 1 to 3 days. Pick out the bacterial colonies that grow faster and have a more typical shape, and then separate and purify them by streaking on the plate for 3 times, then pick out the single colonies in th...

Embodiment 2

[0039] Utilize above-mentioned Pseudomonas (Pseudomonas sp.) ECO-1 bacterial strain to produce the method for bifunctional enzyme preparation, the steps are as follows:

[0040] (1) Streak Pseudomonas sp. ECO-1 strain from -80°C refrigerator on LB solid medium, and culture it upside down at 28°C for 1 day;

[0041] The LB solid medium has the following components per liter:

[0042] Peptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, water to 1L, pH natural;

[0043] (2) Pick a single colony of ECO-1 into LB liquid medium, and culture it on a shaker for 1 day at a temperature of 28°C and a rotation speed of 200 rpm to obtain a seed solution;

[0044] Described LB liquid culture medium, per liter composition is as follows:

[0045] Peptone 10g, yeast extract 5g, sodium chloride 10g, water to 1L, pH natural;

[0046] (3) Inoculate the seed solution prepared in step (2) in the inorganic salt medium at a volume percentage of 10%, and expand the cultivation for 5 days ...

experiment example 1

[0054] Experimental Example 1. Analysis of the Degradation Ability of Bifunctional Enzymes to PCBs

[0055] Mix pentachlorobiphenyl PCB114 substrate, bifunctional enzyme, and PBS buffer at a concentration of 25 mg / L at a ratio of 1:5:19 (volume ratio), react at 30°C and pH 7.0 for 12 hours, and add 10 mL of n-hexane Extracted with alkane for 3 times, the extract was taken, and the degradation rate of pentachlorobiphenyl was detected by GC-MS method, and the degradation rate of PCB114 could reach 65.7%.

[0056] In the comparative example, the intracellular enzyme of the bacterial strain Pseudomonas putida ATCC 12633, PBS buffer solution, and the pentachlorobiphenyl PCB114 substrate with a concentration of 25mg / L were mixed in a ratio of 5:19:1 (volume ratio), and then mixed at 30°C and pH7.0 After reacting for 12 hours, add 10 mL of n-hexane to extract 3 times, take the extract, and use GC-MS method to detect the degradation rate of pentachlorobiphenyl, PCB114 is not degraded....

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Abstract

The invention relates to pseudomonas sp. as well as a preparation method and application of a dual-functional enzyme preparation of the pseudomonas sp. A pseudomonas sp. ECO-1 strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on March 31, 2017, with the preservation number of CGMCC No. 13960. The pseudomonas sp. ECO-1 strain is obtained by separating from POPs (Persistent Organic Pollutants) polluted soil for the first time; the dual-functional enzyme preparation capable of efficiently degrading polychlorinated biphenyl and atrazine is prepared by utilizing the strain for the first time; especially, the dual-functional enzyme preparation has remarkable degradation activity on the polychlorinated biphenyl which is difficulty degraded under an aerobic condition and the performance is completely different from functions of existing pseudomonas sp. and enzyme preparations thereof; the pseudomonas sp. has a large-scale production and application prospect.

Description

technical field [0001] The invention relates to a preparation method and application of a pseudomonas strain and a bifunctional enzyme preparation thereof, belonging to the field of environmental biotechnology. Background technique [0002] Persistent organic pollutants (POPs) are a class of persistent, semi-volatile, long-distance migration, bioaccumulative and highly toxic organic pollutants in the environment. They are widely distributed and do great harm to the human body. Among them, industrial chemicals polychlorinated biphenyls (PCBs) are the first batch of POPs included in the international Stockholm Convention. The higher the degree of chlorination, the greater the toxicity and the more difficult it is to decompose. The organochlorine pesticide atrazine is a triazine herbicide with a long half-life. Because of its strong mobility and serious pollution to soil, aquatic ecosystems and drinking water sources, it has been nominated as a new POPs substance by the academi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A62D3/02C12R1/38C12R1/40A62D101/22
CPCA62D3/02C12N1/20A62D2101/22C12R2001/38C12N1/205C12R2001/40C12N2500/74
Inventor 季蕾王加宁张强陈贯虹王磊磊傅晓文宋繁永李天元
Owner ECOLOGY INST SHANDONG ACAD OF SCI
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