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Esterase gene and encoded protein as well as application

A technology of gene encoding and esterase, applied in the fields of recombinant plasmids, esterase genes, genetically engineered strains, and esterases

Pending Publication Date: 2019-09-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, esterases with relatively high degradation efficiency that can simultaneously degrade 14 kinds of PAEs with branched chain carbon numbers of 1-8 in Table 1 have not been reported yet.

Method used

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  • Esterase gene and encoded protein as well as application
  • Esterase gene and encoded protein as well as application
  • Esterase gene and encoded protein as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]The cloning of embodiment 1 esterase EST-T2 gene, the construction of expression vector

[0029] (a) According to the genome sequence of the Bacillus subtilis endophyte HB-T2, clone the degrading PAE esterase gene EST-T2, the nucleotide sequence of which is shown in Seq ID NO.1.

[0030] The sequences of the upstream and downstream primers are respectively shown in SEQ ID NO.3 and SEQ ID NO.4, and the two primers respectively have BamHI and HindIII restriction sites to amplify the complete sequence of the EST-T2 gene.

[0031] Amplification system: (Beijing Qingke Xinye Biotechnology Co., Ltd. kit (TSE003):: 2×TSINGKE TM MasterMix25ul; 10uMprimerF / R1ul; DNA2 Oupto50ul); Amplification program: pre-denaturation at 94°C for 5min; cycle parameters: denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 90s; run for 30 cycles; extension at 72°C for 10min, stabilization at 4°C for 1min, and the reaction ended.

[0032] The purified PCR product (Beijing...

Embodiment 2

[0037] Induced expression and purification of embodiment 2 esterase EST-T2 protein

[0038] Inoculate the recombinant strain BL21 (pET32b-EST-T2) into 4ml LB liquid medium containing ampicillin (50μg / mL), culture overnight at 37°C with shaking; the next day, transfer to 100mL LB liquid medium at a ratio of 1:100 , cultured with shaking at 37°C until OD 600 When the value is 0.5, add IPTG to a final concentration of 0.2mmol / L, and induce at 30°C for 24h; centrifuge the expressed Escherichia coli at 8000rpm at 4°C, discard the supernatant, add 20mL of PBS buffer to resuspend the bacteria and resuspend The centrifugation was repeated 3 times, and finally 20 mL of PBS buffer was added to resuspend the bacteria, and they were crushed with an ultrasonic breaker in an ice-water bath (ultrasonic conditions were: ultrasonic time 2 s, intermittent time 8 s, and the total working time was set to 10 min). Complete the preparation of the crude enzyme solution. SDS-PAGE electrophoresis wa...

Embodiment 3

[0040] Example 3 Analysis of Esterase EST-T2 Degradation PAEs Spectrum

[0041] Get 1mL of the crude enzyme solution made in Example 2 and add it into 4mL of PBS buffer to form a 5mL reaction system, add a mixed solution of 14 kinds of PAEs in Table 1 dissolved in acetonitrile so that the concentration of various PAEs in the reaction system is 10mg / L , and then reacted in a constant temperature water bath at 37°C for 1 h, and verified the content of PAEs by GC-MS. The empty crude enzyme solution of pET32b was used as the blank control, and each treatment was repeated 3 times.

[0042] The extraction and analysis method of 14 kinds of PAEs: Add the same amount of acetonitrile to the reaction system to be tested, shake and extract on a shaker at 150 r / min for 40 minutes, take out and add 15gNaCl, vortex for 2min to make layers, draw 1mL supernatant into 10mL glass In a centrifuge tube, blow dry with nitrogen, dilute to 1 mL with chromatographically pure acetonitrile, dilute 10 ...

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Abstract

The invention provides an esterase gene with broad-spectrum degradation and an encoded protein thereof as well as application. The esterase is derived from the esterase gene of bacillus subtilis, and can be applied to degrade various phthalic acids. The invention further constructs a genetically engineered strain and plasmid capable of expressing the esterase, thereby realizing the heterologous expression of the enzyme in escherichia coli, and laying a good foundation for industrial production of the enzyme.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to an esterase gene, an esterase, a recombinant plasmid, a genetic engineering bacterial strain and applications thereof. Background technique [0002] Both esterases and lipases are carboxylic acid hydrolases classified into 8 families. Esterases can hydrolyze the ester bonds of short-chain fatty acids with less than 10 carbon atoms, while lipases can hydrolyze the ester bonds of long-chain fatty acids with more than 10 carbon atoms. Lipase and esterase are important industrial enzyme preparations, which can catalyze hydrolysis, transesterification, esterification and other reactions, and are widely used in oil processing, food, medicine, daily chemical and other industries. Esterases and lipases from different sources have different catalytic characteristics and catalytic activities. Among them, the large-scale production of esterases and lipases with esterification or tran...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21A62D3/02A62D101/28C12R1/19
CPCC12N9/16C12N15/70A62D3/02A62D2101/28
Inventor 万群徐文君李易芯余向阳
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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