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Laccase gene as well as encoded protein and application thereof

A technology of laccase and gene, applied in the field of laccase gene and its encoded protein and its application

Inactive Publication Date: 2013-03-27
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on bacterial laccases, and more bacterial laccases with high decolorization ability and their genes need to be further explored and studied.

Method used

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  • Laccase gene as well as encoded protein and application thereof
  • Laccase gene as well as encoded protein and application thereof
  • Laccase gene as well as encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Bacillus vallismortis fmb-103 cells (CGMCC No.6198) were collected by centrifugation, and the genomic DNA of Bacillus vallismortis fmb-103 was extracted with Shanghai Sangon Genomic DNA Extraction Kit.

[0032] Design two primers based on the registered Bacillus laccase gene (No.GU972592.1) in the Genebank database:

[0033] Upstream primer F-1: 5'-ATGACACTTGAAAAATTTGTGGATGC-3' (SEQ ID NO.3);

[0034] Downstream primer R-1: 5'-TTATTTATGGGGATCAGTTATATC-3' (SEQ ID NO.4);

[0035] In a 50 μl system, the final concentration of each primer is 1 μM, the final concentration of dNTPs is 0.2 mM, fmb-103 strain genomic DNA 10 ng, 2 U Pfu DNA polymerase. The amplification program was 94°C for 3min; 30×(94°C for 40s, 53°C for 50s, 72°C for 90s); 72°C for 10min. Agarose gel electrophoresis, gel cutting, recovery by Shanghai Sangon kit, connection of the recovered PCR product with TaKaRapMD19-simple-T vector, transformation of E.coli DH5α, and spreading on LB containing IPTG, X-gal...

Embodiment 2

[0037] According to the obtained laccase gene sequence, two primers were designed, the upstream primer plus the SacI recognition sequence, and the downstream primer plus the XhoI recognition sequence (the underlined part is the restriction enzyme recognition sequence):

[0038] Upstream primer F-2: 5′-CGC GAGCTC ATGACACTTGAAAAATTTGTGGATGC-3' (SEQ ID NO. 5),

[0039] Downstream primer R-2: 5′-CCG CTCGAG TTATTTATGGGGATCAGTTATATC-3' (SEQ ID NO. 6),

[0040] Add each component according to the following PCR system to amplify the LOX gene:

[0041]

[0042] The PCR program is: 94°C for 3min; 30×(94°C for 40s; 53°C for 50s; 72°C for 90s); 72°C for 10min.

[0043] Purify the PCR product with Shanghai Sangon PCR Product Purification Kit, add SacI, XhoI double enzyme digestion, inactivation, ethanol precipitation, ddH 2 O was redissolved, ligated with an appropriate amount of vector pET-23a digested with the same restriction enzymes, and transformed into Escherichia coli DH5α. ...

Embodiment 3

[0045] Transform the expression plasmid pET-23a-fmb-L103 containing fmb-L103 into Escherichia coli expression host strain BL21(DE3)pLysS, culture at 37°C for 10-11 hours, pick small colonies, insert into 50ml LB liquid culture containing ampicillin Base, 70-90rpm, 30°C culture overnight, according to the volume ratio of 1:40, take the seed solution and add it to 100ml LB liquid medium containing ampicillin, shake at 180rpm at 35°C for 2-3 hours until the OD600 is about 0.6, add IPTG (final Concentration 100μg / ml) induced. After 1.5 hours, the cells were collected by centrifugation. The bacteria were crushed, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of recombinant Bacillus vallismortis laccase (fmb-rL103).

[0046] The laccase activity was determined using the 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) method with slight modifications: the total volume of the reaction system was 3mL, including 2.45mL 0.2mol / L pH ...

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Abstract

The invention belongs to the field of biotechnology, and discloses a laccase gene as well as an encoded protein and an application thereof. The open reading frame sequence of the laccase gene fmb-L103 is shown in SEQ ID NO. 1. The amino acid sequence of the protein encoded by the laccase gene fmb-L103 disclosed by the invention is shown in SEQ ID NO. 2. The laccase gene fmb-L103 disclosed by the invention is applied to decoloration for triphenlmethane dyes. The inventor clones from the selected dead bacillus vallismortis fmb-L103 with a spore laccase activity to obtain a novel prokaryotic laccase gene fmb-L103, and realizes the heterologous efficient expression of escherichia colli expression host bacteria. The recombinase fmb-L103 can be used for efficiently catalyzing the degradation of the triphenlmethane dyes of malachite green, brilliant green and aniline blue.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a laccase gene, its encoded protein and its application. Background technique [0002] Triphenylmethane dye is a kind of polyphenyl ring compound, which is widely used and used in a large amount, and its output is listed as the third major dye. Some of the dyes and intermediate degradation products have "three effects". Moreover, some of these dyes are difficult to degrade, which often leads to unsatisfactory treatment effects of conventional biological treatment systems. Effective dye wastewater treatment methods and technologies are an important guarantee for improving the ecological environment, ensuring food safety and maintaining human health. The biological method is to screen or construct specific microbial strains with high performance for the decolorization of dye wastewater. It is an economical, effective and suitable technology for large-scale wastewater treatment. [000...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/10C12N15/70C02F3/00
Inventor 陆兆新张充吕凤霞别小妹
Owner NANJING AGRICULTURAL UNIVERSITY
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