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DEHP (di-2-ethylhexyl phthalate) hydrolase, gene and application of hydrolase

A kind of phthalate, hydrolase technology

Active Publication Date: 2019-10-25
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some studies have obtained DEHP degrading enzymes by protein precipitation, but the related genes and protein sequences have not been reported, hindering further research

Method used

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  • DEHP (di-2-ethylhexyl phthalate) hydrolase, gene and application of hydrolase
  • DEHP (di-2-ethylhexyl phthalate) hydrolase, gene and application of hydrolase
  • DEHP (di-2-ethylhexyl phthalate) hydrolase, gene and application of hydrolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Gene Cloning of DEHP Hydrolase GoEst15

[0047] According to the open reading frame of DEHP hydrolase GoEst15, the upstream and downstream primers were designed, and the genomic DNA of Gordonia polyisoprenivorans was used as a template for PCR amplification.

[0048] The designed upstream and downstream primers are as follows:

[0049] Upstream primer SEQ ID No.3:

[0050] 5'-G GAATTC ATGGGAGTTCCGAACACC-3';

[0051] Downstream primer SEQ ID No.4:

[0052] 5'-CCC AAGCTT TCAGACGTCGAGCGCGGC-3';

[0053] Wherein, the underlined part of the upstream primer is the cleavage site of the restriction endonuclease EcoR I, and the underlined part of the downstream primer is the cleavage site of the restriction endonuclease Hind III.

[0054] The PCR system was: 2×Taq PCR MasterMix 25 μL, 0.5 μL each of upstream primer and downstream primer (100 μM), 1 μL genomic DNA of Gordonia polyisoprenivorans (100 ng / μL), and 23 μL wxya 2 O. The PCR amplification program is:...

Embodiment 2

[0055] Example 2 Preparation of DEHP hydrolase GoEst15 recombinant expression plasmid and recombinant expression transformant

[0056] The target DNA fragment of DEHP hydrolase obtained by PCR amplification in Example 1 and the pET28a empty plasmid were double-digested with restriction endonucleases EcoR I and Hind III at the same time, then purified by agarose gel electrophoresis, and recovered by a DNA kit. The recovered enzyme-digested target fragment and empty vector were separated at T 4 Under the action of DNA ligase, they were ligated at 4°C for 16 hours to obtain the recombinant plasmid pET28a-GoEst15.

[0057] Transform the resulting recombinant plasmid into E.coli DH5α, spread it on an LB medium plate containing 50 μg / mL kanamycin, and incubate at 37°C for 12 hours. Perform colony PCR verification on the grown colonies, and pick and amplify successfully A positive clone with a target band of about 1620bp in length was obtained. After sequencing and verification, the ...

Embodiment 3

[0058] Example 3 Induced expression of DEHP hydrolase GoEst15

[0059] The recombinant expression transformant E.coli BL21(DE3) / pET28a-GoEst15 obtained in Example 2 was inoculated into LB medium containing 50 μg / mL kanamycin, shaken at 37°C for 12 hours, and then pressed Inoculate 1% (v / v) of the inoculum into a 250mL Erlenmeyer flask containing 50mL of LB medium (containing 50μg / mL kanamycin), put it in a shaker, and culture it with shaking at 37°C and 200rpm. OD of liquid 600 When the temperature reached 0.6, IPTG was added to a final concentration of 1 mmol / L for induction. After induction at 16°C for 24 hours, the culture medium was centrifuged at 12,000 rpm to collect cell pellets and washed with saline to obtain resting cells. Suspend the resting cells obtained by the above method in 10 mL of potassium phosphate buffer (100 mM, pH 7.0), ultrasonically break in an ice-water bath, and centrifuge to collect the supernatant, which is the crude enzyme solution of recombinant...

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Abstract

The invention relates to Gordonia polyisoprenivorans, DEHP (di-2-ethylhexyl phthalate) hydrolase expressed by Gordonia polyisoprenivorans, a coding gene and amino acid sequence of the DEHP hydrolase,a recombinant expression vector and recombinant expression transformant containing the gene sequence, a recombinase of the DEHP hydrolase, a preparation method of the recombinase, and a method of degrading PAEs (phthalate esters) by the recombinase. Compared with the prior art, the DEHP hydrolase has the advantages of wide substrate spectrum, good catalytic effect, mild reaction condition, good environmental friendliness and the like; therefore, the DEHP hydrolase is well applicable to biological remediation of soil and biodegradation of pollutants.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a Gordonia polyisoprenivorans and its expressed DEHP hydrolase, the coding gene and amino acid sequence of the enzyme, and the recombinant expression containing the coding gene The vector, the recombinant expression transformant, and the preparation method of the recombinant enzyme also relate to the application of the recombinant enzyme as a catalyst to degrade phthalate plasticizers. Background technique [0002] Since the 1930s, phthalates (PAEs) have been widely used as plasticizers in the production and processing of various plastic products, including food packaging, blood containers, industrial pipes, cable sheathing, agricultural plastic films As well as vehicle plastic products, etc., covering important industries such as daily necessities, medical care, construction and automobiles. According to statistics, in 1975, the global output of phthalates wa...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/16C12N15/55C12N15/70C12P7/02C12P7/62C12R1/01
CPCC12N9/16C12P7/02C12P7/62C12N1/205C12R2001/01
Inventor 黄晗张晓彦白云鹏徐殿胜
Owner EAST CHINA UNIV OF SCI & TECH
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