Preparation method and application of sludge petroleum degrading complex enzyme

A technology of petroleum degradation and compound enzymes, applied in the field of soil treatment, can solve the problems of poor prospects and high application costs, and achieve the effects of high-efficiency oil pollution treatment capacity, low production costs, and broad application prospects.

Active Publication Date: 2018-09-04
ECOLOGY INST SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above technical solutions can be applied to oil-contaminated water bodies and soil remediation processes, since the oil concentration used is up to 10g/L, the actual concentration of pol

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Acinetobacter calcoacetate ( Acinetobacter calcoaceticus ) cells were cultured and prepared as follows:

[0054] a. Acinetobacter calcium acetate ( Acinetobacter calcoaceticus ) was transferred to LB medium according to the mass ratio of 2%, and the seeds were cultured at 30°C and 160rpm for 15 hours to obtain the seed solution;

[0055] b. Transfer the seed solution prepared in step a to LB medium with an inoculum size of 4% to 5% by mass, and expand the culture at 30°C and 160rpm for 15 hours to obtain a bacterial solution;

[0056] C, the bacterium liquid that step b is made is through centrifugation, collects precipitation, makes Acinetobacter calcoacetate ( Acinetobacter calcoaceticus ) cells.

Embodiment 2

[0058] formate dehydrogenase Cb The preparation steps of FDH are as follows:

[0059] (i) Construction of genetically engineered strain Escherichia coli E. coli BL21- fdh ; The construction method is as follows:

[0060] Amplified from Candida boidinii ( Candida boidinii ) formate dehydrogenase gene fdh , the gene sequence is shown in SEQ ID NO.2, the upstream amplification primer sequence is shown in SEQ ID NO.3, the amplified formate dehydrogenase gene fdh Linked to E. coli expression vector pET28a(+), constructed to carry fdh Gene recombinant expression vector pET28a(+)- fdh , transform the host bacteria Escherichia coli BL21 (DE3), pick the transformants, and screen to obtain recombinant Escherichia coli expressing formate dehydrogenase E. coli BL21- fdh ; Refer to the instructions for use of Escherichia coli expression vector pET28a(+) for specific step conditions;

[0061] (ii) The genetically engineered strain Escherichia coli constructed in step (i...

Embodiment 3

[0070] A kind of preparation method of sludge oil degradation compound enzyme, the steps are as follows:

[0071] (1) Acinetobacter calcium acetate prepared in Example 1 ( Acinetobacter calcoaceticus ) cells were crushed, centrifuged at 5000 r / min for 2 min, and the supernatant was taken to obtain the petroleum degrading enzyme solution 21 # ;

[0072] The cells are broken, and the steps are as follows:

[0073] Acinetobacter calcoacetate ( Acinetobacter calcoaceticus ) cells were mixed evenly with phosphate buffer solution at pH 7.5 at a mass volume ratio of 1:20, unit g / ml; under 320 W ultrasonic conditions, the cells were disrupted by intermittent ultrasonic treatment for 17 min, each time ultrasonic Time 2s, intermittent time 2s;

[0074] (2) The petroleum degradation enzyme liquid 21 prepared in step (1) # It is mixed with formate dehydrogenase at a ratio of protein mass ratio of 1:4 to prepare sludge oil degradation compound enzyme.

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Abstract

The invention relates to a preparation method and application of a sludge petroleum degrading complex enzyme. The preparation method comprises the following steps: (1), performing cell disruption on acinetobacter calcoaceticus, centrifuging, and taking a supernatant to obtain a petroleum degrading enzyme solution 21#; (2), mixing the petroleum degrading enzyme solution 21# and formate dehydrogenase to obtain the sludge petroleum degrading complex enzyme. For the first time, after a petroleum-degrading enzyme system derived from acinetobacter calcoaceticus is mixed with the formate dehydrogenase in proportion, the sludge petroleum degrading complex enzyme can be applied to degradation and repair of petroleum in high-concentration petroleum-contaminated sludge, so that immobilization of adsorbents such as diatomite is not required, and the petroleum in the sludge can be reduced in a short time. The sludge petroleum degrading complex enzyme has high-efficiency petroleum contamination treatment capacity, has low production cost, and has a wide application prospect.

Description

technical field [0001] The invention relates to a preparation method and application of a compound enzyme for degrading oil sludge and petroleum, and belongs to the technical field of soil treatment. Background technique [0002] During the production, storage, transportation, refining, processing and use of petroleum, due to accidents, abnormal operation and maintenance, etc., there will be spillage and discharge of petroleum hydrocarbons. For example: blowout accidents during oilfield development; leakage accidents of oil pipelines and oil storage tanks; leakage accidents of oil tankers and tankers; wax removal in oil wells and maintenance of oilfield ground equipment; maintenance of oil refining and petrochemical production equipment, etc. Petroleum hydrocarbons are spilled in large quantities and should be recovered as much as possible. However, recovery is difficult in some cases. Even if efforts are made to recover, some of them will remain and cause pollution to the e...

Claims

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Application Information

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IPC IPC(8): C12N1/20C02F11/02C12N9/02C12N9/00C12N15/70C12R1/01
CPCC02F11/02C12N1/20C12N9/0008C12N15/70C12Y102/01002C02F3/342C02F3/344C02F2101/32C12N9/00C12Y102/00B09C1/10C12N9/96C12N15/66C12Y102/02001
Inventor 季蕾张强王加宁傅晓文宋繁永李天元陈贯虹郭书海
Owner ECOLOGY INST SHANDONG ACAD OF SCI
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