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TFLFM (Fucoidan Lyase from Fucobacter marina) as well as preparation method and application thereof

A technology of fucoidan sulfate and lyase, which is applied in the field of genetic engineering to achieve high-efficiency expression

Active Publication Date: 2018-09-14
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there are relatively few reports on the research on sulfated fucoidan degrading enzymes. There is no commercialized sulfated fucoidan degrading enzyme preparation on the market, and there is no sulfated fucoidan oligosaccharide product (KusaykinMI) prepared by biological methods on the market. , et al., Glycobiology, 2016, 26, 3-12)

Method used

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  • TFLFM (Fucoidan Lyase from Fucobacter marina) as well as preparation method and application thereof
  • TFLFM (Fucoidan Lyase from Fucobacter marina) as well as preparation method and application thereof
  • TFLFM (Fucoidan Lyase from Fucobacter marina) as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Construction of complete sulfated fucoidan lyase FLFM expression strain

[0031] The present invention refers to the gene encoding sulfated fucoidan lyase (GenBank No: AAO00510.1), through codon optimization, entrusts Bioengineering (Shanghai) Co., Ltd. to synthesize the coded sulfated fucoidan lyase (excluding signal peptide) The gene has a total of 2022 bases, the nucleotide sequence is shown in SEQ ID NO.1, the cloning vector is pET28a, the cloning sites are NdeI and XhoI, the carrier resistance is kanamycin (Kan), and the optimized species is E.coli ( figure 1 ). Escherichia coli DH5α carrying the expression plasmid pET28a-FLFM was cultured, the plasmid was extracted using the Plasmid Mini Kit I kit, and then the expression plasmid was introduced into competent Escherichia coli BL21

[0032](DE3), a recombinant strain was obtained. The amino acid sequence is shown as SEQ ID NO.2, which contains 673 amino acids, and the predicted protein molecular weight...

Embodiment 2

[0057] Example 2 Expression and detection of complete sulfated fucoidan lyase FLFM

[0058] (1) Prepare LB medium containing Kan resistance: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride, sterilize at 120°C for 20min, cool to room temperature and add Kan to make the final concentration 50μg / L mL.

[0059] (2) Inoculate the recombinant strain obtained in Example 1 onto a solid LB medium containing Kan resistance, culture overnight at 37°C, pick a single colony and inoculate it into 5 mL of liquid LB culture containing Kan resistance, at 37°C, After culturing on a shaker at 200rmp for 24 hours, inoculate the above bacterial solution into 500ml of liquid LB culture containing Kan resistance, and culture at 37°C and 200rmp bed to OD 600nm When =0.6, add 0.5mM IPTG, induce expression at 16°C, 200rmp for 24 hours, centrifuge at 5000rmp, and collect the bacteria.

[0060] (3) A small amount of bacteria was taken for SDS-PAGE analysis, and the expression of the target pr...

Embodiment 3

[0061] Example 3 Construction of N-terminal truncated sulfated fucoidan lyase TFLFM expression strain

[0062] With F1(5'-CGGCGC CATATG CAGACTACTACCGTATACAG-3') and R1(5'-ACGGGC CTCGAG TTAATCGGAGTCGCAGTCAATCGC-3') was used as a primer pair, and the plasmid pET28a-FLFM was used as a template for PCR, the PCR product and the expression vector pET28a were digested with NdeI and XhoI respectively, the target fragment was recovered, and the gene fragment and the vector fragment were ligated to obtain the plasmid pET28a-TFFLFM ( figure 1 ). Escherichia coli DH5α carrying the expression plasmid pET28a-TFFLFM was cultured, the plasmid was extracted using the Plasmid Mini Kit I kit, and then the expression plasmid was introduced into competent Escherichia coli BL21(DE3) to obtain a recombinant strain. The nucleotide sequence is shown in SEQ ID NO.3, the amino acid sequence is shown in SEQ ID NO.4, which contains 447 amino acids, and the predicted protein molecular weight is 48.0kD...

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Abstract

The invention relates to the field of gene engineering, in particular relates to TFLFM (Fucoidan Lyase from Fucobacter marina) as well as a preparation method and application thereof. By shortening anamino acid sequence of TFLFM, the high-efficient expression can be realized. A strain used in the invention is escherichia coli BL21(DE3), and a gene coding the shortened TFLFM is introduced, so thatthe recombinant Escherichia coli of the high-efficiently expressed TFLFM can be obtained, and the enzymatic activity is verified by virtue of in-vitro experiment.

Description

technical field [0001] The invention relates to the field of genetic engineering, and mainly relates to a sulfated fucoidan lyase TFLFM and a preparation method and application thereof. Background technique [0002] Fucoidan sulfate or fucoidan sulfate is a water-soluble heteropolysaccharide with high molecular weight, mainly found in brown algae (such as kelp, fucus, etc.) and some marine invertebrates (such as sea cucumbers, sea urchins, etc.) middle. It is mainly composed of sulfated L-fucose (2 / 3 / 4 hydroxyl sulfate of fucose), some also contain galactose, mannose, uronic acid, glucose, rhamnose, xylose and other mono Components such as sugars and acetyl groups (Ale MT, et al., Mar. Drugs, 2011, 9, 2106-2130). The structure of fucoidan sulfate derived from brown algae is very complex, including the position and quantity of sulfation and acetylation, as well as branched chain structure, etc. Only the average or partial structure of fucoidan sulfate is relatively clear. ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P19/04
CPCC12N9/88C12N15/70C12P19/04
Inventor 杜昱光李建军贾培媛任立世
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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