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Enzyme preparation for treating sewage containing estrogen pollutants

An enzyme preparation and sewage technology, applied in the field of enzymology, can solve the problems of difficulty in directly treating neutral and alkaline sewage, high price, and difficulty in production.

Active Publication Date: 2019-10-15
SHANGHAI HUA SURNAME PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biodegradation technology is one of the effective ways to solve this pollution problem. The most commonly used enzyme is peroxidase, such as horseradish peroxide, manganese peroxidase, lignin peroxidase, etc. Horseradish peroxidase The main problem of lignin peroxidase is that it is difficult to produce and the price is high, while manganese peroxidase and lignin peroxidase have the problem of low enzyme activity above neutral pH value, so it is difficult to directly treat common neutral and alkaline sewage. However, adjusting the pH of a large amount of sewage or waiting for it to cool requires a lot of time, space and cost

Method used

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  • Enzyme preparation for treating sewage containing estrogen pollutants
  • Enzyme preparation for treating sewage containing estrogen pollutants
  • Enzyme preparation for treating sewage containing estrogen pollutants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Obtaining of Lip-15

[0022] Error-prone PCR and mutant enzyme library construction

[0023] The SG-LIP nucleotide sequence in CN201410462409.5 (SEQ ID NO.1 in CN201410462409.5) was synthesized, ligated, and transformed into Escherichia coli. Sequence the plasmid of a single colony to confirm that the sequence contained in the plasmid is correct and use it as a template.

[0024] The plasmid obtained in the previous step was used as a template, and Agilent's GeneMorph II randommutagenesis kit was used to perform three rounds of error-prone PCR. The PCR product is recovered and connected to the carrier. Competent cells were prepared, transformed into Escherichia coli, positive clones were screened, and positive clones were collected to form a mutant enzyme library.

[0025] mutant screening

[0026] Inoculate the positive clones on the culture plate to induce culture for 120 hours, take the supernatant to measure the enzyme activity, the enzyme activity at ...

Embodiment 2

[0027] Example 2 Expression and purification of SG-LIP and Lip-15 in Escherichia coli

[0028] Inoculate wild-type SG-LIP and Lip-15 positive clone engineering bacteria into LB medium in a 50ml flask, add 100uM IPTG to induce culture for 24 hours, centrifuge to obtain crude enzyme solution, and then pass dialysis, concentration, GST purification kit The purified wild-type SG-LIP and Lip-15 were purified, and SDS-PAGE showed a basic single band with a molecular weight of about 45kD. After sequencing, its sequence was determined to be SEQ ID NO.2.

Embodiment 3

[0029] The enzymatic property of embodiment 3 SG-LIP, Lip-15

[0030] The enzyme activity detection of lignin peroxidase adopts the assay method (Tien M, Kirk T K.Lignin peroxidase of Phanerochaete chrysosporium.MethodsEnzymol, 1988,161:238-249) of widely accepted Tien and Kirk in this area, an enzyme Activity unit is defined as the amount of enzyme needed to catalyze the production of 1umol veratraldehyde per minute.

[0031] At room temperature, when the pH is 4.5, the enzyme activities of the crude enzyme solution of SG-LIP and Lip-15 in Example 2 are 158.2U / L and 147.5U / L respectively. The enzyme activity of the crude enzyme solution is close to that.

[0032] Prepare the buffer system (0.25M) of 3.0-7.0 with malonic acid-sodium malonate buffer solution, detect the enzyme activity of SG-LIP, Lip-15 crude enzyme solution at different pHs (three times) according to the above-mentioned method, will SG-LIP and Lip-15 crude enzyme solutions were incubated in a pH 6.0 buffer a...

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Abstract

The invention discloses an enzyme preparation for treating sewage containing estrogen pollutant. The enzyme preparation contains a Streptomyces griseorubens lignin perxidase mutant, wherein the aminosequence of the mutant is SEQ ID NO.2, and the nucleotide sequence of the mutant is SEQ ID NO.1.

Description

technical field [0001] The invention relates to the fields of enzymology and sewage treatment. Specifically, the invention provides a lignin peroxidase mutant of Streptomyces grisea and its application in sewage treatment of estrogen-containing pollutants. Background technique [0002] Lignin peroxidase (LiP) is the main lignin-degrading enzyme in the biodegradation process of lignin, which can form free radicals in lignin polymers, resulting in poor bond stability and destroying lignin macromolecules. . Kent and Ming discovered LiP for the first time in 1983 from the restricted medium of Phanerochaetechrysosporium, a white-rot fungus, and believed that LiP belongs to oxidoreductase and is a heme-containing heme secreted by fungi. Glycosylated extracellular protein peroxidase. Later, it was successively used in Coridus versicolor, Thametes versicolor, Bjerkandera adusta, Phlebia radiata, Pleurotus pulmonanus, and Pycnoporus cinnabarinu. Lignin peroxidase has been found in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/08C02F3/34C02F101/34
Inventor 王翠华
Owner SHANGHAI HUA SURNAME PHARMA