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Alginate lyase and gene and application thereof

An alginate lyase, gene technology, applied in the directions of lyase, application, genetic engineering, etc., can solve the problems of long growth cycle, enzyme inactivation, enzyme activity reduction, etc., and achieve high activity, long-lasting stability, temperature adaptability, etc. strong effect

Active Publication Date: 2019-09-20
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of intracellular enzymes requires mechanical disruption for release from cells, which may result in inactivation or reduced enzyme activity, limiting the large-scale production and application of alginate lyases
However, the operation of Pichia pastoris is complex and its growth cycle is longer

Method used

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  • Alginate lyase and gene and application thereof
  • Alginate lyase and gene and application thereof
  • Alginate lyase and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Alginate lyase Gene acquisition

[0039] (1) Derived from Microvesicles Microbulbifer sp .Isolation and extraction of genomic DNA:

[0040] Take 1.5 mL of the cell culture Microbulbifer sp. (purchased from China Marine Microorganism Culture Collection Management Center) in a sterilized Ep tube, centrifuge at 12000 rpm for 1 min, discard the supernatant, and collect the cells.

[0041] Add 400 μL of lysate solution (40 mM Tris-acetic acid, 20 mM sodium acetate, 1 mM EDTA, 1% SDS, pH 7.8), mix well, and place in a 37°C water bath for 1 h.

[0042] Then add 200 μL of 15 mol / L sodium chloride solution, mix well and centrifuge at 13000 rpm for 15 min.

[0043] Take the supernatant, extract twice with phenol and once with chloroform.

[0044] Add twice the volume of absolute ethanol and 1 / 10 volume of potassium acetate (3 M, pH 8.0), store at -20°C for 1 h, centrifuge at 13,000 rpm for 15 min, discard the supernatant, and wash the precipitate twice with 70% et...

Embodiment 2

[0055] Example 2 Alginate lyase Expression and amplification of coding genes in Escherichia coli

[0056] With the result obtained in embodiment 1-(4) Hind III and Bam H I double-enzyme-digested target gene, and Hind III and Bam H The pET28a (+) plasmid of I double digestion is ligated to obtain the recombinant plasmid pET-28a -Alginate lyase (like figure 1 shown).

[0057] Take 10uL of the constructed plasmid DNA and add it to 100uL of the prepared Escherichia coli BL21(DE3) competent cells. Shake well and place on ice for 30 minutes; heat shock in a water bath at 42°C for 90 seconds; place the centrifuge tube quickly Transfer to ice-water mixture for ice bath for 2min; add 800uL LB medium to each tube, lightly aspirate and disperse with a pipette, and recover on a shaker at 37°C for 1h (80rpm-200rpm); centrifuge at 4000rpm×5min, remove 700uL supernatant , and mix the rest; smear the plate (LB plate, containing 100ug / mL Amp), place it upright at 37°C for 1 hour, ...

Embodiment 3

[0059] Example 3 Construction of Yeast Recombinant Expression Vector

[0060] The result obtained in embodiment 1-(4) Avr II and not I double-enzyme-digested target gene, and Avr II and not I double-digested pPIC9k plasmid connection to obtain recombinant plasmid pPIC9k -Alginate lyase (like figure 1 shown).

[0061] Transfer the recombinant plasmid into E. coli Top10 competent cells were spread on the LB screening plate containing 100 mg / mL Amp and cultured overnight, and the recombinants were picked into liquid LB (containing 100ug / mL Amp), and identified by bacterial liquid PCR, if they were positive recombinants , Sent for sequencing, the sequencing results further prove the correctness of the target gene sequence and insertion site. Select the bacterial solution with the correct inserted gene, follow the extraction steps of the OMEGA Plasmid Mini Kit, extract the recombinant plasmid, and store it at -20°C for later use.

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Abstract

The invention provides alginate lyase and a gene and application thereof and further provides a method of cloning and expression of the alginate lyase in escherichia coli and yeast cells. A nucleotide sequence of the alginate lyase is shown as SEQ ID NO.1, and an amino acid sequence of the alginate lyase is shown as SEQ ID NO.3. A recombinant vector comprising the gene is formed by connection of the alginate lyase gene to escherichia coli plasmids or yeast plasmids. A cell comprising the gene is obtained by conversion of the recombinant vector. The cell of the alginate lyase gene comprises the nucleotide molecule or escherichia coli converted from the recombinant vector or comprises the nucleic acid molecule or Pichia pastoris converted from the recombinant vector.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alginate lyase and its gene and application. [0002] technical background [0003] Alginate lyase is mainly derived from marine organisms, including marine microorganisms, invertebrates, algae, etc. Studies have found that the most alginate lyase is polysaccharide lyase family 7 (PL7). Alginate lyase can degrade alginate into oligosaccharides or oligosaccharides with various activities, which have been well established in bacteria. ground research. In 2018, Jingjing Zhuang et al. reported a new alginate lyase from Vibrio, which belongs to the seventh family of polysaccharide lyases. It contains two consecutive PL7 domains, which can degrade alginate to form di- and tri- -, four-algin oligosaccharides. In 2018, Chune Peng et al. discovered an endo-type alginate lyase belonging to the 7th family of polysaccharide lyases. They used different sugar chains as test substr...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/81C12N1/19C12P19/00C12P19/04C12P19/12C12R1/84
CPCC12N9/88C12N15/815C12P19/00C12P19/04C12P19/12
Inventor 李仁宽叶秀云王圆芳郭炳奇
Owner FUZHOU UNIV
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