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Laccase gene slr1573 derived from synechocystis and application thereof in dye decolorization

A technology of slr1573-xhoi-r and slr1573-nhei-f, which is applied in the biological field and can solve the problems of less sources of algal laccase and the like

Pending Publication Date: 2019-10-01
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of less sources of algae laccase in the prior art, the present invention provides a laccase gene derived from Synechocystis sp.

Method used

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  • Laccase gene slr1573 derived from synechocystis and application thereof in dye decolorization
  • Laccase gene slr1573 derived from synechocystis and application thereof in dye decolorization
  • Laccase gene slr1573 derived from synechocystis and application thereof in dye decolorization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 Synechocystis laccase slr1573 Gene PCR amplification

[0059] According to the registered in the Genebank database slr1573 gene, designed primers slr1573- Nhe I-F / slr1573- xho I-R, amplified using Synechocystis sp. 6803 genomic DNA as a template slr1573 The open reading frame of the laccase gene is 789 bp (see the electrophoresis pattern of the PCR product figure 1 ), the nucleotide sequence is shown in SEQ ID NO.1, the encoded protein sequence is shown in SEQ ID NO.2, and the amplification primers are as follows:

[0060] slr1573- Nhe I-F: 5′-ACTC GCTAGC ATGGGGGACG ATCTCTGGGG -3′ (includes NheI restriction site), (SEQ ID NO.3)

[0061] slr1573- xho I-R: 5′-ACTC CTCGAG TCAGTAACTGACAATGCCAG-3′ (contains XhoI restriction site) (SEQID NO.4)

[0062] PCR reaction conditions: pre-denaturation at 98°C for 2min; denaturation at 98°C for 10s, annealing at 55°C for 15s, extension at 72°C for 15s, after 30 cycles; extension at 72°C for 5min, and...

Embodiment 2

[0064] Example 2 slr1573 Expression vector construction

[0065] Positive clones (including slr1573 fragment) with Nhe I and xho I double digestion, recovery slr1573 Fragment; pET28a (+) plasmid also used Nhe I and xho I restriction endonuclease treatment, recovery of pET28a (+) vector fragments. recycled slr1573 The fragment was ligated with the pET28a(+) vector fragment to obtain pET28a(+)- slr1573 plasmid.

Embodiment 3

[0066] Example 3 Recombinant Synechocystis laccase gene slr1573 Expression in E. coli

[0067] pET28a(+) obtained from implementation 2- slr1573 The plasmid was transformed into Escherichia coli BL21 (DE3) (see the PCR identification map image 3 ), to obtain BL21 / PET28a (+)- slr1573 Recombinant expression strains, optimized culture conditions, obtained culture conditions for high-efficiency expression of recombinant laccase, and Western blot detection of Slr1573 recombinant protein.

[0068] 1. Optimal IPTG induction concentration screening:

[0069] Liquid LB activated strains were cultivated overnight, inoculated in 50ml LB liquid medium at a ratio of 1:50 between strains and culture medium, and cultured at 37°C and 180rpm until OD 600 When ~0.6, add the final concentration of 0.1mM, 0.5mM and 1mM different concentrations of inducer IPTG to induce expression, and screen the optimal IPTG induction concentration (for the induction results, see Figure 4 ).

[0070] 2. S...

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Abstract

The invention relates to a laccase gene slr1573 derived from synechocystis 6803 and application thereof in dye decolorization and belongs to the technical field of biology. The DNA sequence of the laccase gene is shown as SEQ ID NO.1, and an amino acid sequence of an encoded protein is shown as SEQ ID NO.2. A prokaryotic expression vector of the synechocystis laccase gene slr1573 is constructed, an escherichia coli expression strain BL21 (DE3) is transformed, the strain expressing the recombinant Slr1573 is successfully obtained, and the enzymatic property of crude recombinant laccase liquid is detected. The obtained slr1573 laccase gene is efficiently expressed, and the recombinant laccase can efficiently catalyze decolorization of dyes such as malachite green.

Description

technical field [0001] The present invention relates to a kind of Synechocystis Laccase gene of 6803 slr1573 And the application in dye decolorization belongs to the field of biotechnology. Background technique [0002] Laccase is a copper-containing polyphenol oxidase, which can catalyze the oxidation of phenolic and non-phenolic aromatic compounds into water, and is an environmentally friendly enzyme. Laccase catalyzes a wide range of substrates, and the scope of substrate action can be further expanded by forming a laccase-mediator system. [0003] Laccase resources are abundant, which can be divided into three categories according to different sources: plant laccase, animal laccase and microbial laccase (including fungal laccase and bacterial laccase). The earliest discovered laccase is sumac laccase, which comes from animal There are few reports on laccases from fungi, such as white rot fungi, Ascomycotina and Deuteromycotina, which are the main source of laccase. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C02F3/34C12R1/19C02F101/38
CPCC12N9/0061C12Y110/03002C12N15/70C02F3/342C02F2101/308C02F2101/38
Inventor 张燕岳寿松张伟陈高于金慧杨建罗时华王俊艳边斐游银伟钟怀荣朱友峰
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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