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Novel deep-sea low-temperature salt-tolerant esterase and application

An esterase, amino acid technology, applied in the field of genetic engineering, to achieve the effect of excellent enzymatic properties

Active Publication Date: 2016-03-16
SECOND INST OF OCEANOGRAPHY MNR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few authorized low-temperature bacterial esterase patents

Method used

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  • Novel deep-sea low-temperature salt-tolerant esterase and application
  • Novel deep-sea low-temperature salt-tolerant esterase and application
  • Novel deep-sea low-temperature salt-tolerant esterase and application

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Effect test

Embodiment 1

[0021] The acquisition of embodiment 1 esterase gene e10

[0022] Bacteria Croceicoccus marinus E4A9 based on deep-sea sediments T The whole genome, open reading frame prediction and gene annotation results are used to screen lipohydrolase-related genes. The homology between the sequence and the known esterase gene sequence in the database was compared by Blastx (http: / / blast.ncbi.nlm.nih.gov / ). The e10 gene was obtained through database comparison analysis, with a size of 618bp and a base composition of 106A (17.15%), 97T (15.70%), 203C (32.85%) and 212G (34.30%), and its nucleotide sequence is as shown in SEQIDNo: 1 shown. The size of the encoded protein is 205 amino acid residues, and its amino acid sequence is shown in SEQ ID No.2. A homology search was performed on the gene sequence in GenBank, and the highest similarity was the esterase in the same genus Croceicoccusnaphthovorans, with a similarity of 64%. Its registration number in the GenBank database was WP_0478202...

Embodiment 2

[0025] The construction of the recombinant expression plasmid of embodiment 2 esterase gene e10 and recombinant bacterial strain

[0026] The esterase gene e10 obtained in the present invention is cloned into an expression vector to construct a recombinant expression strain. Based on the open reading frame sequence of the esterase gene obtained by ORF analysis of NCBIORFFinder, the upstream primer E10F (5'-TCGC GGATCC GTGGCGGACGGCGAGGC-3', BamHI) and downstream primer E10R (5'-TCCG CTCGAG CTAGAGGTCGTCGATCCTGTC-3', XhoI), PCR amplification confirmed the full-length sequence of the gene. The expression plasmid was constructed by enzyme digestion cloning, that is, the PCR product was double-digested with BamHI and XhoI, and the purified fragment was ligated with the plasmid pSMT3 that had been double-digested with BamHI and XhoI. 2 The transformation method was transformed into E.coliDH5α, and positive clones were screened for kanamycin resistance. A plasmid extraction kit (...

Embodiment 3

[0027] Example 3 Utilize recombinant expression strain to express recombinant esterase gene e10

[0028] Transfer 3ml of the constructed recombinant expression strain to 100ml of LB liquid medium containing 20μg / ml kanamycin and 34μg / ml chloramphenicol, and shake at 37°C until OD 600 When it reaches 0.6, add IPTG with a final concentration of 0.5mM to induce expression, transfer to 25°C and shake at 150r / min for 8h. The bacterial cells were collected by low-temperature centrifugation, resuspended in NTA-10 solution (500 mM sodium chloride, 10 mM imidazole, 20 mM Tris hydrochloric acid, pH 8.0), and subjected to sonication on ice. Centrifuge at low temperature to collect supernatant, use NTA-Ni 2+ The expressed protein was purified by affinity column chromatography. The expressed recombinant protein contains N-terminal 6×Histag, which can be affinity-adsorbed to the layer suction column, and is eluted with different concentrations of imidazole solution gradient, and the eluat...

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Abstract

The invention discloses novel deep-sea low-temperature salt-tolerant esterase E10 and a coding gene and application thereof. The invention relates to the esterase gene e10 coming from deep-sea bacteria Croceicoccus marinus E4A9T, and a nucleotide sequence is shown in SEQ ID No.1. After heterologous expression is conducted on an esterase gene, when a substrate is p-nitro phenol caproate (C6), the catalytic activity is the maximum, and the enzyme activity is 29.4 U / mg. The optimum temperature of esterase catalytic hydrolysis of the esterase E10 ranges from 15 DEG C to 20 DEG C; the activity remains 80 percent or above in 1 mol / L NaCl; under the condition of adding organic solvent of dimethyl sulfoxide, glycerin and isopropanol, the enzyme activity is increased. The esterase has the advantages of being low in temperature, resistant to salt and capable of being applied to industrial production such as chiral drug synthesis, food processing and food flavor improvement, wastewater treatment and washing industry under the low-temperature salt-containing condition.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a low-temperature salt-tolerant esterase derived from deep-sea bacteria, its coding gene and its application. Background technique [0002] Lipohydrolases are a class of enzymes that catalyze the hydrolysis and synthesis of esters. According to the biochemical characteristics and sequence characteristics of lipolytic enzymes, lipolytic enzymes from microorganisms are mainly divided into 8 families. There is a similar amino acid sequence Gly-x-Ser-x-Gly near the active site Ser of most lipolytic enzymes, but the sequence near the active site of the II family is Gly-Asp-Ser-Leu (GDSL), Therefore, this family is also called the GDSL family. Due to the variability of its structure, GDSL family esterases usually have a variety of hydrolysis functions and a wide substrate spectrum. This activity has a wide range of potential applications in industry. [0003] In recent...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N15/81C12N15/85C12N15/70C12N1/21C12N1/19C12N5/10C12P7/62
CPCC12N9/18C12P7/62C12Y301/01
Inventor 许学伟霍颖异王春生孟凡旭王昭崔恒林
Owner SECOND INST OF OCEANOGRAPHY MNR
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