JP2022513548A - Preparation method and application of microbacterium paraoxydance, its broad-spectrum polychlorinated biphenyl enzyme preparation, and its application. - Google Patents

Abstract

The present invention relates to a method for preparing a polychlorinated biphenyl enzyme preparation having a broad spectrum of microbacterium paraoxydance, and an application thereof. Microbacterium paraoxydans ECO-2 was entrusted to the Ordinary Microbial Center of the China Microbial Species Conservation Management Committee on June 01, 2018, and the contract number of the bacterial species was CGMCC No. It is 15836. The intracellular complex enzyme obtained by inducing and culturing the microbacterium paraoxydance ECO-2 strain on a single substrate which is biphenyl is a low-chlorination / perchlorination of PCB28, PCB101 and / or PCB114 under aerobic conditions. The enzyme preparation is clearly different from the conventionally known ones that can decompose the converted PCBs, has a wide decomposition spectrum, and can decompose only a single component in a single induction culture, and the future potential of the application can be expected. [Selection diagram] None

Description

The present invention belongs to the technical field of microorganisms with respect to microbacterium paraoxydance, a method for preparing a polychlorinated biphenyl enzyme preparation having a wide spectrum thereof, and an application thereof.

Persistent organic pollutants, polychlorinated biphenyls (PCBs), are highly toxic, bioaccumulative and long-distance migratory, have a long half-life, and severely pollute soil, aquatic ecosystems and drinking water sources, resulting in malformations. And cause cancer. Both the Taiwan Yusho incident and the Japan Kanemi Yusho incident caused by PCBs have caused serious loss of life and property. Due to its thermal and chemical stability, flame retardancy, insulation and oxidation resistance, PCBs are widely applied in the chemical, electric, electronic and mechanical industries and are currently used in China's PCBs and their contaminants. The existing amount is large, and a large amount of PCBs contaminated land is generated due to electronic waste and industrial land relocation. In 2015, the Ministry of Environmental Protection announced the National Environmental Protection Standard (HJ743-2015) "Gas Chromatography for Measuring Polychlorinated Biphenyls in Soil and Sediments-Mass Analysis Method" to measure PCBs in soil and sediments. Standardized the method. There is already an urgent need to treat PCBs pollution in a timely and effective manner, protect the environment and ensure human health. PCBs isomers are diverse, PCBs with a coplanar structure have toxicity similar to dioxin, and PCBs as indicators are monomers specified by the United Nations GEMS / FOOD as indicators for monitoring the status of PCB contamination. There, trichlorobiphenyl PCB28 and pentachlorobiphenyl PCB101, which are the main PCBs pollutants in the environment, are both typical compounds thereof.

Polychlorinated biphenyls (PCBs) have thermal and chemical stability, flame retardancy, insulation and oxidation resistance, so they are widely applied in the chemical industry, electric power, electronics and machinery industries, and their applications are. Mainly insulating oil, flame retardant, heat conductive agent, hydraulic oil, plasticizer, railway transformer, mining equipment, electromagnetic equipment, carbonless carbide paper, pigment, wax additive, dust remover, pesticide additive, lubrication Includes agents, cutting oils, sealants and blockers. In China, the cumulative production of polychlorinated biphenyls over the years is close to 10,000 tons, and although the production was basically stopped in China in the early 1980s, power capacitors including PCBs from some developed countries, Due to the import of power transformers and, after the 90's, foreign countries imported polychlorinated biphenyls into China mainly through electronic waste, the existing amount of PCBs and their pollutants in China is still very high and industrial land relocation. Therefore, there is a large amount of PCB contaminated land. PCBs invade soil and water environment due to waste discharge, oil storage tank leaks, volatilization, dryness, wet sedimentation, etc., causing serious pollution to soil, aquatic ecosystems and drinking water sources, and against PCBs. The problem of secondary and persistent contamination of PCBs, coupled with poor management and improper treatment and storage, is quite serious.

Patent Document 1; Chinese Patent 107287134A (Application No. 201710508652.X) discloses a Pseudomonas sp. ECO-1 strain. Deposited at the center, the deposit number is CGMCC No. It is 13960. The present invention is the first to isolate a Pseudomonas sp. ECO-1 strain from POPs-contaminated soil, and to use this strain to produce for the first time a bifunctional enzyme preparation capable of efficiently degrading polychlorinated biphenyls and atladines. However, this enzyme preparation has remarkable degrading activity against highly chlorinated polychlorinated biphenyls, which are particularly difficult to decompose under aerobic conditions, and has completely different functions from the conventionally known Pseudomonas sp. And its enzyme preparations. Is different, and it is expected to be applied to mass production.

However, the strains still fail to meet the actual treatment needs in terms of treatment speed and types of polychlorinated biphenyls that can be treated, and rapid repair against PCBs complex contamination under aerobic conditions is the focus of current research. It has become.

Chinese Patent 107287134A (Application No. 201710508652.X)

The present invention provides a method for preparing a microbacterium paraoxydance, a broad-spectrum polychlorinated biphenyl enzyme preparation thereof, and an application for defects in the prior art.

The technical proposal of the present invention is as follows.

On June 01, 2018, it was entrusted to the Ordinary Microbial Center of the China Microbial Bacterial Species Preservation Management Committee (Address: No. 1, No. 1, Hokushin West Road, Chaoyang District, Beijing, No. 3, Institute of Microbial Science, Chinese Academy of Sciences), and the contract number of the bacterial species was CGMCC No. Microbacterium paraoxydans ECO-2, which is 15836.

As shown in FIG. 1, the purely cultured monoclonal form of this strain is that the colonies are yellow, the edges and the surface are smooth, and the colonies are circular.

The method for culturing the above strains is
The step (1) of obtaining an activated strain by drawing a microbacterium paraoxydans ECO-2 strain on a solid activation medium and activating and culturing the strain.
The activated strain obtained in step (1) is inoculated into a liquid medium and cultured with shaking to obtain a seed solution.
This includes the step (3) of transferring the seed solution obtained in step (2) to an expanded medium at a volume of 2% to 10% and expanding the culture to obtain a bacterial solution.

According to the present invention, preferably, in the step (1), the solid activation medium is an LB solid medium, and the composition is as follows.
Peptone is 10 g, yeast extract is 5 g, sodium chloride is 10 g, and agar is 20 g. The pH is adjusted to 1 L with water and has a pH in a natural state.

According to the present invention, preferably, in the step (1), the activation condition is inverted culture at 28 to 37 ° C. for 1 to 2 days.

According to the present invention, preferably, the liquid medium in the step (2) and the expanded medium in the step (3) are both LB liquid media, and the composition is as follows.
It is 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride, and the pH is adjusted to 1 L with water and has a pH in a natural state.

According to the present invention, the shaking culture condition in the step (2) is preferably 28 to 37 ° C. and a rotation speed of 100 to 200 rotations / minute for 1 to 2 days. ..

According to the present invention, preferably, the expansion culture condition in the step (3) is to expand culture at 28 to 37 ° C. and 20 to 70% of dissolved oxygen for 1 to 2 days.

Application of the above Microbacterium paraoxydans ECO-2 in the preparation of enzyme preparations for repairing polychlorinated biphenyl-contaminated soil.

The above application
Microbacterium paraoxydans ECO-2 bacterial solution in 1-10% volume percent, inorganic salt induction medium containing biphenyl at a concentration of 0.3-0.8 g / L. To obtain a induced Microbacterium paraoxydans ECO-2 bacterial solution by inoculating the medium with a temperature of 28 to 37 ° C. and a rotation rate of 100 to 200 rpm for 3 to 5 days. Step (i) and
The induced Microbacterium paraoxydans ECO-2 bacterial solution obtained in step (i) is solid-liquid separated, the bacterial cells are suspended in a phosphate buffer solution, cell disruption is performed, and further 4 to 4 This includes the step (ii) of obtaining an enzyme preparation by solid-liquid separation under the condition of 25 ° C. and collecting the supernatant.

According to the present invention, preferably, in the step (i), the composition of the inorganic salt medium per liter is
Potassium dihydrogen phosphate 0.5 g, disodium hydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, calcium chloride 0.1 g, sodium chloride 0.2 g, ammonium sulfate 1.0 g, peptone 2.0 g, pH 7.0 Is.

According to the present invention, preferably, in the step (ii), the solid-liquid separation is performed by centrifugation for 2 to 10 minutes under the condition of 3000 to 10000 rpm.

According to the present invention, preferably, in the step (ii), the cell disruption employs a high-pressure homogenized cell disruption method.

According to the present invention, preferably, in the step (ii), the phosphate buffer solution is a phosphate buffer solution having a pH of 5.0 to 8.0, and the amount of the phosphate buffer solution used is preferably 5 to 50. Double capacity.

Application of the above enzyme preparation in the repair of polychlorinated biphenyl-contaminated soil.

According to the present invention, preferably the polychlorinated biphenyl is 2,4,4'-trichlorobiphenyl (PCB28), 2,2', 4,5,5'-pentachlorobiphenyl (PCB101) and / or 2 , 3, 4, 4', 5-pentachlorobiphenyl (PCB114).

1. In the present invention, the Microbacterium paraoxydans ECO-2 strain is disclosed for the first time, and the intracellular complex enzyme obtained by inductive culture with a single substrate which is biphenyl is used under aerobic conditions. , PCB28, PCB101 and / or PCB114 and the like can degrade low-chlorinated / hyperchlorinated PCBs, and enzyme preparations have a wide degradation spectrum and can only degrade a single component in a single induction culture. Obviously different from the ones, it is more environmentally friendly and more convenient to handle on a large scale than the physical and chemical treatment methods, has a higher decomposition rate and shortens the repair cycle than the microbial method. The decomposition efficiency is improved, and the future of application can be expected.
2. Wide-spectrum polychlorinated biphenyl enzyme preparations prepared using Microbacterium paraoxydans ECO-2 are PCB 114 with coplanar structure, and trichlorobiphenyl PCB28 and pentachlorobiphenyl PCB101, which are indicators of environmental pollution. Degradation activity is high for both, the decomposition rate for pentachlorobiphenyl PCB101 reaches 100%, the range of application is wide, and it is not necessary to use perchlorinated PCBs, which are more toxic at the time of preparation, for induction. Therefore, the preparation process is simple and is expected to be applied to mass production.

Photograph of a purely cultured monoclonal form of the Microbacterium paraoxydans ECO-2 strain

Hereinafter, the technical proposal of the present invention will be further described in Examples, but the scope of the invention is not limited thereto.

Origin of Organism Microbacterium paraoxydans ECO-2 was established on June 01, 2018, at the China Microbial Species Conservation Management Committee Ordinary Microbial Center (Address: No. 1 Hokushin West Road, Chaoyang District, Beijing). It was entrusted to No. 3 Institute of Microbiology, Chinese Academy of Sciences), and the contract number of the bacterial species was CGMCC No. It is 15836.

Medium LB solid medium has the following composition per liter.
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, 1 L of water, pH in the natural state
In the LB liquid medium, the composition per liter is as follows.
Peptone 10g, yeast extract 5g, sodium chloride 10g, water to 1L, pH in natural state
In the inorganic salt medium, the composition per liter is as follows.
Potassium dihydrogen phosphate 0.5 g, disodium hydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, calcium chloride 0.1 g, sodium chloride 0.2 g, ammonium sulfate 1.0 g, peptone 2.0 g, 1 L with water Constant volume, pH 7.0.

Example 1
POPs-contaminated soil basin leachates were prepared and diluted to five concentration gradients of 10 ^-1 , ^10-2 , 10 ^-3 , 10 ^-4 , and ^10-5 , respectively. The diluted bacterial suspension was applied to a solid medium containing biphenyl and cultured at 30 ° C. for 1 to 3 days. Bacterial colonies with high growth rate and typical morphology are selected, separated and purified by three plate strokes, and then a single colony is placed in an inorganic salt liquid medium at 30 ° C. and 150 rpm. After culturing in 1 for 3 days, 0.5 mL of glycerin was added to 1.5 mL of the culture and mixed uniformly, and then stored for a long time in a refrigerator at -80 ° C.

The solid medium to which the bacterial suspension is applied is an LB solid medium, and the composition is as follows.
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, 1 L of water, pH in the natural state.

The composition of the inorganic salt liquid medium for culturing a single colony is as follows.
Potassium dihydrogen phosphate 0.5 g, disodium hydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, calcium chloride 0.1 g, sodium chloride 0.2 g, ammonium sulfate 1.0 g, peptone 2.0 g, 1 L with water Constant volume, pH 7.0.

The strains obtained above were used as polychlorinated biphenyl PCB28 (2,4,4'-trichlorobiphenyl), PCB101 (2,2', 4,5,5'-pentachlorobiphenyl) and PCB114 at a concentration of 25 mg / L, respectively. Inoculate an inorganic salt liquid medium containing (2,3,4,4', 5-pentachlorobiphenyl), incubate at 150 rpm for 72 hours at 30 ° C., observe the turbidity of the bacterial solution, and observe the bacteria. Absorption values at 600 nm were detected for the suspension. Enzyme-producing strains were selected based on the above indicators. The strain having the highest absorption value was taken in LB solid medium, cultured, and stored as ECO-2.

The monoclonal was sent to Qingdao ▲ Chin ▼ Kazushi Biotechnology Co., Ltd. for sequencing, and when detected, the 16S rDNA sequence was contained at 1357 bp, and the nucleotide sequence was SEQ ID NO. As shown in 1, when the bacterial species were identified, it was Microbacterium paraoxydans, which was named Microbacterium paraoxydans ECO-2, and was named Microbacterium paraoxydans ECO-2. The committee was entrusted to the Ordinary Microbial Center (Address: No. 1, No. 3, Hokushin West Road, Chaoyang District, Beijing, Microbial Research Institute, Chinese Academy of Sciences), and the consignment number of the bacterial species was changed to CGMCC No. It was set to 15836.

The identification of the bacterial species is as follows.
Sample: Bacterial solution Bacterial genome DNA extraction kit: Amane Biochemical Technology (Beijing) Co., Ltd., DP302
TAE buffer (50 × 1 L): Tris 242 g, glacial acetic acid 57.1 ml, Na2EDTA. 2H2O 37.2g, water added up to 1L Agarose: BIOWET, AGAROSE G-10
2 × Pfu PCR MasterMix, D2000DNA Marker, Nucleic Acid Dye, Loading Buffer, etc .: Amane Biochemical Technology (Beijing) Co., Ltd. DNA Purification and Recovery Kit: Amane Biochemical Technology (Beijing) Co., Ltd., DP214
Consumables such as centrifuge tubes and pipette tips: Gene Era Biotech (GEB), USA

Primer: Qingdao ▲ Chin ▼ Synthesized by Kazushi Azusa Biotechnology Co., Ltd. According to the synthetic instruction manual, ddH ₂ O was added to prepare a 10 μM solution.
1. Genomic DNA extraction was performed according to the DP302 kit.
2. PCR amplification

2.3 PCR cycle parameters Preliminary denaturation: 94 ° C, 3 min, denaturation 94 ° C, 30 s, annealing 55, 30 s, elongation, 72 ° C, 1.5 min (total 35 cycles), extension 72 ° C, 10 min; storage at 4 ° C ..

3. Agarose gel electrophoresis detection A 1.0% agarose gel was prepared, and the voltage was 18 V / cm and the electrophoresis time was 20 min for electrophoresis.

4. Purification and recovery Agarose gel was recovered for the target fragment using a general agarose gel DNA recovery kit, and the recovered products were sequenced by Qingdao Chin ▼ Kazushi Biotechnology Co., Ltd.

Example 2
The steps of the method for preparing a broad-spectrum polychlorinated biphenyl degrading enzyme preparation using the Microbacterium paraoxydans ECO-2 of Example 1 are as follows.
(1) Microbacterium paraoxydans ECO-2 was drawn on an LB solid medium and subjected to inverted activation culture at 35 ° C. for 2 days to obtain an activated strain.
(2) The activated strain obtained in step (1) was inoculated into an LB liquid medium and cultured with shaking at 35 ° C. and a rotation speed of 200 rpm for 2 days to obtain a seed solution.
(3) The seed solution obtained in step (2) was transferred to an inorganic salt medium containing 0.5 g / L of biphenyl at a volume of 10%, and the conditions were 35 ° C. and 70% dissolved oxygen for 2 days. The cells were expanded and cultured to obtain a bacterial solution.
(4) The bacterial solution obtained in step (3) is centrifuged at 3000 rpm for 10 minutes to collect the bacterial cells, and the cells are suspended in a 30-fold volume of a phosphate buffer solution having a pH of 7.0. After turbidity and high-pressure homogenized cell disruption, centrifugation was performed at 4 ° C. and 3000 rpm for 2 minutes, and the supernatant was collected to obtain a polychlorinated biphenyl degrading enzyme preparation having a wide spectrum.

Comparative Example 1
When an enzyme preparation was prepared by the method of Example 2 using Microbacterium paraoxydans strain MA25 having the most similar genetic relationship and cultured in the inorganic salt medium of step (3), the bacterial solution became turbid. However, when the absorption value at 600 nm was detected for the bacterial suspension, the value at OD _{600 nm} was zero. Therefore, in the inorganic salt medium of step (3), the microbacterium. It was revealed that the paraoxydance Microbacterium paraoxydans strain MA25 cannot survive.

Comparative Example 2
(Pseudomonas sp.) Using the ECO-1 strain, an enzyme preparation was prepared by the method of Example 2. When the cells were cultured in the inorganic salt medium of step (3), turbidity of the bacterial solution was observed, and when the absorption value of the bacterial suspension at 600 nm was detected, the value of OD _{600 nm} was 1.05. From this, it was clarified that Pseudomonas sp. ECO-1 can survive in the inorganic salt medium of step (3).

Experimental example Each of pentachlorobiphenyl PCB114, PCB101, and trichlorobiphenyl PCB28 having a concentration of 25 mg / L was mixed with a broad-spectrum polychlorinated biphenyl enzyme preparation and PBS buffer at a ratio of 1: 5: 19 (volume ratio). The reaction was carried out at 30 ° C. and pH 7.0 for 10h, 13h and 10h, respectively, 10 mL of n-hexane was added and the mixture was extracted three times, and the decomposition rate of the above polychlorinated biphenyl was detected in the extract by the GC-MS method.
As a result of the detection, the broad-spectrum polychlorinated biphenyl degrading enzyme preparation obtained in Example 2 had a decomposition rate of trichlorobiphenyl PCB28 reaching 90% within 10 hours under aerobic conditions, and decomposition to dioxin-like pentachlorobiphenyl PCB114. The rate reached 57.1% within 10 hours, and the decomposition rate with respect to the index pentachlorobiphenyl PCB101 reached 100% within 13 hours.

In Comparative Example 2, the decomposition rate reached 60.3% within 10 hours only for the dioxin-like pentachlorobiphenyl PCB114, and the decomposition rate was zero for the others.

As can be seen from the above data, the Microbacterium paraoxydans ECO-2 of the present invention produces a broad spectrum polychlorinated biphenyl enzyme preparation for PCB114, PCB101 and PCB28 when induced with a single substrate. It has high application value as compared with the conventional known enzyme preparations which can only decompose a single substance when induced by a single substrate.

Claims (10)

Bacterial species entrusted to the China Microbacterium Species Preservation Management Committee Ordinary Microbial Center (Address: No. 1, No. 3, Hokushin West Road, Chaoyang District, Beijing, China Institute of Microbiology) on June 01, 2018 (Contract number: CGMCC) No. 15836), Microbacterium paraoxydans ECO-2. The method for culturing Microbacterium paraoxydans ECO-2 according to claim 1.
The step (1) of obtaining an activated strain by drawing a microbacterium paraoxydans ECO-2 strain on a solid activation medium and activating and culturing the strain.
The activated strain obtained in step (1) is inoculated into a liquid medium and cultured with shaking to obtain a seed solution.
A culture method comprising the step (3) of transferring the seed solution obtained in step (2) to an expanded medium at a volume percentage of 2% to 10% and expanding the culture to obtain a bacterial solution. .. In the step (1), the solid activation medium is an LB solid medium, and the composition is:
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, the pH was adjusted to 1 L with water, and the pH was in a natural state.
Preferably, the culture method according to claim 2, wherein in the step (1), the activation condition is inverted culture at 28 to 37 ° C. for 1 to 2 days. The liquid medium in step (2) and the expanded medium in step (3) are both LB liquid media, and the composition is as follows.
It is 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride.
The second aspect of the present invention is preferably that the shaking culture condition in the step (2) is 28 to 37 ° C. and a rotation speed of 100 to 200 rotations / minute for 1 to 2 days. Culture method. The culture method according to claim 2, wherein the expansion culture condition in the step (3) is expansion culture at 28 to 37 ° C. and a condition of 20 to 70% dissolved oxygen for 1 to 2 days. An application of the Microbacterium paraoxydans ECO-2 according to claim 1.
An application characterized by being an application in the preparation of an enzyme preparation for repairing polychlorinated biphenyl-contaminated soil. Microbacterium paraoxydans ECO-2 bacterial solution in 1-10% volume percent, inorganic salt induction medium containing biphenyl at a concentration of 0.3-0.8 g / L. To obtain a induced Microbacterium paraoxydans ECO-2 bacterial solution by inoculating the medium with a temperature of 28 to 37 ° C. and a rotation rate of 100 to 200 rpm for 3 to 5 days. Step (i) and
The induced Microbacterium paraoxydans ECO-2 bacterial solution obtained in step (i) was solid-liquid separated, the bacterial cells were suspended in a phosphate buffer solution, cell disruption was performed, and further 4 to 4 to The application according to claim 6, comprising the step (ii) of obtaining an enzyme preparation by solid-liquid separation under the condition of 25 ° C. and collecting the supernatant liquid. In step (i), the composition of the inorganic salt medium per liter is
Potassium dihydrogen phosphate 0.5 g, disodium hydrogen phosphate 0.5 g, magnesium sulfate 0.2 g, calcium chloride 0.1 g, sodium chloride 0.2 g, ammonium sulfate 1.0 g, peptone 2.0 g, pH 7.0 And
Preferably, in step (ii), the solid-liquid separation is centrifugation at 3000 to 10000 rpm for 2 to 10 minutes.
Preferably, in the step (ii), the cell disruption is to adopt a high-pressure homogenized cell disruption method.
Preferably, in the step (ii), the phosphate buffer is a phosphate buffer having a pH of 5.0 to 8.0, and preferably, the amount of the phosphate buffer used is 5 to 50 times the volume. The application described in 7. It is an application of the enzyme preparation obtained in claim 8.
An application characterized by being a response in the restoration of polychlorinated biphenyl contaminated soil. The polychlorinated biphenyls are 2,4,4'-trichlorobiphenyl, 2,2', 4,5,5'-pentachlorobiphenyl and / or 2,3,4,4', 5-pentachlorobiphenyl. The application according to claim 9.

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