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Marine microorganism Arthrobacter sp. YJ34 dextranase producing gene and recombinant engineering bacterium thereof

A dextranase, marine microorganism technology, applied in genetic engineering, microorganisms, plant genetic improvement and other directions, can solve the problems of high production cost, long-term inability of bacterial cells, repeated use, poor stability, etc.

Inactive Publication Date: 2019-04-26
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the enzyme is an induced enzyme, after the enzyme activity reaches its peak in the logarithmic phase of growth, the enzyme activity decreases rapidly and the stability is poor. The bacteria cannot be reused for a long time, resulting in high production costs.

Method used

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  • Marine microorganism Arthrobacter sp. YJ34 dextranase producing gene and recombinant engineering bacterium thereof
  • Marine microorganism Arthrobacter sp. YJ34 dextranase producing gene and recombinant engineering bacterium thereof
  • Marine microorganism Arthrobacter sp. YJ34 dextranase producing gene and recombinant engineering bacterium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1, Marine microorganism Arthrobacter ( Arthrobacter sp.) YJ34 dextranase gene cloning experiment:

[0061] 1 Total DNA extraction of Arthrobacter marine

[0062] Bacterial culture

[0063] Inoculate activated bacteria in 5.0mL liquid medium (yeast powder 1.0, peptone 5.0, MgSO 4 0.4, NaCl 4.0, dextran 20000 10.0, 1L distilled water, pH 7.5, autoclaved at 121°C for 30 minutes. ), 37°C, 220 rpm, shaker for 12 hours to obtain bacterial culture solution.

[0064] (2) Bacteria collection

[0065] Take the above bacterial culture solution into an Eppendorf tube, centrifuge at 12000g for 10 min at room temperature, discard the supernatant, and resuspend the pellet in 1 mL TE (pH 8.0).

[0066] (3) Bacteria lysis

[0067] First add 6μL of 50mg / mL lysozyme and act at 37℃ for 2h. Then add 50μL of 2M NaCl, 110μL of 10% SDS (sodium dodecyl sulfonate), 3μL of 20mg / mL proteinase K, and act at 50°C for 3h to fully lyse the bacteria to obtain a lysate.

[0068] (4) Extraction

[0069] Tr...

Embodiment 2

[0097] Example 2, Marine microorganism Arthrobacter ( Arthrobacter sp.) Expression of YJ34 dextranase gene in Escherichia coli:

[0098] 1. Construction of expression vector pET-21a(+)-Dextranase

[0099] PMD-18T-Dextranase and the empty vector pET-28a(+) were digested by Nde1 and EcoR1 respectively, and the gel was recovered and ligated overnight at 16°C for transformation E.coli JM109, pick the positive recombinants on the LB plate containing kanamycin resistance, extract the recombinant plasmid and perform PCR and double enzyme digestion verification, the positive clone is pET-28a(+)-Dextranase (such as image 3 Shown).

[0100] 2. Induced expression

[0101] Take 1μL of pET-21a(+)-Dextranase, transfer to E.coli BL21(DE3), take 100μL to spread on the LB plate containing ampicillin (100ug / mL), incubate at 37℃ for 10h, pick a single colony to 5mL of LB Incubate at 37°C for 10 hours in the culture medium, take 1mL of culture broth to 100mL of LB medium, culture at 37°C to OD600 of...

Embodiment 3

[0109] Example 3, Experiment of dextranase gene recombinant engineering bacteria:

[0110] 1. Materials and testing methods

[0111] Arthrobacter marine ( Arthrobacter sp.) YJ34, obtained by our laboratory's own separation and screening.

[0112] Enzyme activity determination method: add 100 μL enzyme solution to 100 μL 3% dextran 70,000 (50 mmol / L, pH 6.5 PBS buffer solution dissolving preparation), 37°C water bath for 30 min, add 200 μL DNS to the reaction solution to stop For reaction, add 3ml of distilled water to dilute after 5min in boiling water bath, read the absorbance value at 520nm in the microplate reader after spotting the plate.

[0113] A 250 mL Erlenmeyer flask containing 50 mL of culture medium was inserted into Arthrobacter marina YJ34, cultured at 30°C for 18-20 hours until OD600 = 1.0, and the genomic DNA was extracted using the Bacterial Genome Extraction Kit of Hangzhou Baosai Biology Company. Scan for sequencing.

[0114] 2. Construction of recombinant bacteria...

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Abstract

The invention provides a marine microorganism Arthrobacter sp. YJ34 dextranase producing gene. An enzyme gene sequence is 1923 bp in total length, ATG is taken as an initial codon, TAG is taken as a terminal codon, and GC base content is 59.23 percent. The invention further discloses a dextranase gene recombinant engineering bacterim obtained through cloning of the dextranase gene and efficient expression in escherichia coli. The invention belongs to the technical field of enzymology and enzyme engineering. The recombinant engineering bacterium is built through gene cloning and sequencing, anda purified recombinant enzyme is obtained by induced expression and purification of a recombinant enzyme. The enzyme activity and enzyme stability are improved greatly. The optimal reaction temperature of the enzyme is 55 DEG C as determined according to the enzymatic property, and the optimal acting pH is 7.5.

Description

Technical field [0001] The present invention relates to a marine Arthrobacter ( Arthrobacter sp.) The dextranase gene produced by YJ34 and the recombinant engineering bacteria obtained by cloning of the dextranase gene produced by the marine microorganism Arthrobacter YJ34 and high-efficiency expression in coliform species belong to the field of enzymology and enzyme engineering technology. Background technique [0002] Arthrobacter marine ( Arthrobacter sp.) can grow on cheap raw materials such as corn flour, syrup, molasses, soybean meal, bran and other industrial and agricultural wastes, easy to achieve large-scale cultivation, and has dextranase activity. However, since the enzyme is an inducible enzyme, after the enzyme activity reaches its peak in the logarithmic phase, the enzyme activity declines quickly, and the stability is poor. The bacteria cannot be reused for a long time, resulting in high production costs. In order to improve enzyme activity and stability, it is ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/46C12N15/70C12N1/21C12R1/19
CPCC12N9/2454C12N15/70C12Y302/01011
Inventor 王淑军刘红飞任伟吕明生房耀维刘姝
Owner HUAIHAI INST OF TECH
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