Method for extracting catechol 2,3-dioxygenase from microalgae and purifying catechol 2,3-dioxygenase

A catechol and dioxygenase technology, applied in biochemical equipment and methods, redox enzymes, enzymes, etc., to achieve the effect of simple operation method, easy operation conditions and high purity

Inactive Publication Date: 2018-03-30
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that there are many bacteria capable of degrading aromatic compounds in nature, and can extract and detect C23O from the bacteria, but there are few reports on C23O in microalgae

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Microbiological identification and separation of water samples collected from the scene of the oil spill accident, from which chlorella was screened out, and the chlorella was enriched and cultivated: the medium was boiled with seawater for 15 minutes, and nutrient salts were added after suction filtration , the concentration of each component in the nutrient salt is NH 4 Cl 0.2g / L, K 2 HPO 4 0.05g / L and ferric citrate 0.01g / L, after 15 days, the algae liquid was obtained;

[0026] (2) Suction filter the algae solution obtained in step (1), wrap the filter membrane enriched with algae with tinfoil, place it in liquid nitrogen to cool for 30 seconds, take it out, add 4g of quartz sand in a mortar at 4°C Grinding at 1.2mmol / L pH 7.0 phosphate buffer solution to wash out the algae liquid, centrifuging at 10000r / min for 15min in a centrifuge, and taking the supernatant; the precipitated part was washed twice with phosphate buffer solution, combined and centrifuged th...

Embodiment 2

[0030] (1) Microbiological identification and separation of water samples collected from the scene of the oil spill accident, from which chlorella was screened out, and the chlorella was enriched and cultivated: the medium was boiled with seawater for 10 minutes, and nutrient salt was added after suction filtration , the concentration of each component in the nutrient salt is NH 4 Cl 0.1g / L, K 2 HPO 4 0.1g / L and ferric citrate 0.005g / L, after 10 days the algae liquid was obtained;

[0031] (2) Suction filter the algae solution obtained in step (1), wrap the filter membrane enriched with algae with tinfoil, place it in liquid nitrogen to cool for 90 seconds, take it out, add 3g of quartz sand in a mortar at 0°C Grinding at 1.5mmol / L, pH 7.5 phosphate buffer solution to wash out the algae liquid, centrifuged at 10000r / min for 15min in a centrifuge, and took the supernatant; the precipitated part was washed twice with phosphate buffer solution and combined for 3 The supernata...

Embodiment 3

[0035] (1) Microbiological identification and separation of water samples collected from the scene of the oil spill accident, from which chlorella was screened out, and the chlorella was enriched and cultivated: the medium was boiled with seawater for 20 minutes, and nutrient salts were added after suction filtration , the concentration of each component in the nutrient salt is NH 4Cl 0.3g / L, K 2 HPO 4 0.075g / L and ferric citrate 0.02g / L, after 7 days the algae liquid was obtained;

[0036] (2) Suction filter the algae solution obtained in step (1), wrap the filter membrane enriched with algae with tinfoil, place it in liquid nitrogen to cool for 60 seconds, take it out, add 5g of quartz sand in a mortar at 2°C Grind at 1.0mmol / L, pH 7.5 phosphate buffer solution to wash out the algae liquid, centrifuge at 10,000r / min for 15min, and take the supernatant; wash the precipitated part twice with the above phosphate buffer solution, and combine The supernatant of 3 centrifugati...

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Abstract

The invention relates to the technical field of environment protection, in particular to a method for extracting catechol 2,3-dioxygenase from microalgae and purifying catechol 2,3-dioxygenase. The method comprises the following steps of (1) performing microalgae enriching culture; (2) performing suction filtration, washing and resuspension to obtain a coarse enzyme solution A; (3) performing graded precipitation and overnight dialysis to obtain a coarse enzyme solution B; (4) performing gradient elution and overnight dialysis to obtain catechol 2,3-dioxygenase solution. The detection of the catechol 2,3-dioxygenase is expanded to the microalgae for the first time; the operation method is simple and easy; the operation conditions can be easily met; the economic performance is realized. Theefficient arene degradation rate is realized; the catechol 2,3-dioxygenase is further extracted and purified; the purity is high; the degradation effect on the hydrocarbon is obvious.

Description

technical field [0001] The invention relates to the technical field of environmental protection, in particular to a method for extracting and purifying catechol 2,3-dioxygenase from microalgae. Background technique [0002] In recent years, the environmental pollution caused by marine oil spills has increasingly become the focus of people's attention. Among hydrocarbon pollutants, aromatic compounds account for a large proportion. Aromatic compounds are a class of refractory organic compounds, and have a great toxic effect on the human body, can cause cancer and have neurotoxicity. Generally speaking, almost all aromatic compounds are first degraded into catechol during the degradation process, and then catechol is cleaved into muconic acid semialdehyde or muconate by the action of ortho or meta-dioxygenase. Conic acid, thereby breaking the benzene ring. [0003] As an important intermediate product in the degradation process of aromatic compounds, catechol is catalyzed b...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N9/02
CPCC12N9/0069C12Y113/11001
Inventor陈庆国李晶晶刘梅陶玲国青青穆军
OwnerZHEJIANG OCEAN UNIV