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Carboxylesterase gene, recombinant plasmid, recombinant engineered bacterium, encoded protein and application

A technology of recombinant engineering bacteria and protein encoding, applied in the field of bioengineering, can solve the problems of obtaining heterologous expression, rarely obtaining functional proteins, and unable to express proteins

Active Publication Date: 2019-05-17
夏盛(上海)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Streptomyces protein expression often requires protein post-translational modification processes such as glycosylation, so when performing heterologous expression in Escherichia coli (Escherichia coli), due to the lack of a complete protein post-translational modification system in the E. The G+C content of the genome sequence of the mold TK24 is as high as 72.2%, resulting in the protein from Streptomyces often not being expressed or existing in the form of inclusion bodies in Escherichia coli, and active functional proteins are rarely obtained
The locus SLIV_RS20080 on the chromosome of Streptomyces lividans TK24 cannot be heterologously expressed in E. coli due to the high GC content of the Streptomyces genome

Method used

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  • Carboxylesterase gene, recombinant plasmid, recombinant engineered bacterium, encoded protein and application
  • Carboxylesterase gene, recombinant plasmid, recombinant engineered bacterium, encoded protein and application
  • Carboxylesterase gene, recombinant plasmid, recombinant engineered bacterium, encoded protein and application

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Effect test

Embodiment 1

[0036] Optimization, Synthesis and Sequence Analysis of Carboxylesterase Gene estC

[0037]Streptomyces lividans TK24 locus (Locus tag) SLIV_RS20080 is the esterase gene estC, and the name of the protein encoded by this nucleotide sequence is α / β hydrolase (alpha / beta hydrolase), namely carboxylesterase, protein The product (Protein product) number (Accession) is WP_003975294, and the protein accession number in GenBank is AIJ14960. The nucleotide sequence of Streptomyces lividans TK24 esterase gene estC on the chromosome has a start point (Start) of 4442794, a stop point (Stop) of 4443699, a length of 906 bp, and 96 A bases, accounting for 10.6%; T bases are 109, accounting for 12%; C bases are 338, accounting for 37.3%; G bases are 363, accounting for 40.1%; GC content is relatively high, accounting for 77.4%. Due to the high GC content of the esterase gene estC and the complex protein post-translational modification process of Streptomyces, the heterologous expression of t...

Embodiment 2

[0040] Construction of recombinant plasmids and recombinant engineering bacteria

[0041] The artificially synthesized optimized carboxylesterase gene estC' was connected with the plasmid expression vector pET28b carrying the Kan resistance gene to obtain the recombinant plasmid pET28b-est C'. The connection condition is to react overnight at 16°C, and the connection system is:

[0042]

[0043] Mix 10 μL of the above ligation reaction solution with 100 μL of LE.coli Rosetta (DE3) competent cells, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, then immediately ice-bath for 5 minutes, add 500 μL LB medium to resume culture for 1 hour, and construct recombinant engineered bacteria Rosetta(DE3)pLysS / pET28b-estC', the engineered strain has Kan and Cam resistance, and can be screened by LB medium with Kan and Cam resistance.

Embodiment 3

[0045] Expression of carboxylesterase EstC

[0046] Pick the recombinant engineered bacteria from the above-mentioned transformed plates, then inoculate them in a test tube containing 5mL LB liquid medium, shake and cultivate overnight at 37°C, 225rpm; Shaped flask, 37 ° C, 225 rpm shaking culture. Observe the growth of the bacteria during the period, and cultivate to OD 600 When the concentration is 0.4-0.6, add IPTG with a final concentration of 0.01-0.5mM, 180rpm, shake at 16°C-25°C at low temperature, and induce expression for 20-24h. Thereafter, the cells were collected by centrifugation at 5000 rpm for 5 min at 4°C, and ultrasonically disrupted to obtain carboxylesterase EstC.

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Abstract

The invention discloses a thermostable carboxylesterase gene estC', a recombinant plasmid, a recombinant engineered bacterium, an encoded protein and application thereof. The recombinant engineered bacterium Rosetta(DE3)pLysS / pET28b-estC' can achieve heterologous expression of EstC at 16-25 DEG C and 0.01-0.5 mM IPTG; the carboxylesterase EstC has high thermal stability and has no obvious enzyme activity change after placed at 55 DEG C for 180h, and the enzyme activity is still above 80% after incubation at 100 DEG C for 6h. EstC has a high-efficiency degradation effect on organophosphorus pesticide chlorpyrifos. When the concentration of the chlorpyrifos is as high as 65mg / L, 0.5 U esterase EstC can degrade the concentrate by 80% at 45 DEG C for 14 h. The carboxylesterase gene estC', therecombinant plasmid, the recombinant engineered bacterium, the encoded protein and the application can provide services for industrial applications in the fields of pesticide degradation and environmental treatment, especially when high temperature catalysis is required, the carboxylesterase is more suitable.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a heat-resistant carboxylesterase gene, a recombinant plasmid, a recombinant engineered bacterium and its encoded protein, and an application in pesticide degradation. Background technique [0002] Carboxyl esterases (EC 3.1.1.3, carboxyl ester hydrolases) are a large class of enzymes that prefer to catalyze the hydrolysis and formation of ester bonds of substrates with small acyl chain lengths (≤10), and have excellent chemoselectivity , regioselectivity and stereoselectivity, and can be applied in the fields of pesticide degradation, environmental governance and pharmaceutical synthesis. Organophosphorus pesticides have been widely used in agricultural production, but they also bring problems such as environmental pollution and pesticide residues. Compared with the traditional chemical method of degrading pesticides, carboxylesterase can specifically catalyze the cleavage of este...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/70C12N1/21C12N9/20A62D3/02C12R1/19A62D101/04A62D101/26
Inventor 王宝娟吴爽高鹏王鹏朱国萍师雪芹常欣熊声麒杨刚汪勇王剑飞
Owner 夏盛(上海)生物科技有限公司
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