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Cerrena unicolor derived laccase and gene and application thereof

A tooth hairy fungus, source technology, applied in the direction of application, genetic engineering, plant genetic improvement, etc., can solve the problems of limiting the application value of strains, hindering the application of laccase, and long enzyme production cycle, etc., to achieve good economic and social benefits, The effect of short enzyme production cycle and short production time

Active Publication Date: 2020-01-03
FUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, most fungal laccases generally have the problems of long enzyme production cycle, low enzyme production and instability, such as Panus tigrinus The highest enzyme production was 2.2 U / mL when cultured in the optimized medium for 11 days, Lentinus The highest enzyme production of sp. was 58 U / mL when cultured for 16 days, Cerrna unicolor The highest enzyme activity of IBB fermentation was 152 U / mL on the 14th day of fermentation, and the highest enzyme activity of 123 U / mL was obtained on the 15th day of fermentation after another strain of Dendrobium 5.1011 studied in our laboratory was optimized. Application value is one of the main reasons hindering the industrial application of laccase

Method used

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  • Cerrena unicolor derived laccase and gene and application thereof
  • Cerrena unicolor derived laccase and gene and application thereof
  • Cerrena unicolor derived laccase and gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: the fermentative production of laccase

[0065] Fermentative production of LacB laccase from Dendrochaetes monochrome ( Cerrna unicolor) The secondary seed culture of the 87613 strain was inoculated into the fermentation medium at an inoculum volume of 8% (v / v), with a liquid volume of 60 mL / 250 mL, and fermented at 30 °C and 200 rpm.

[0066] Dendrobium monochrome ( Cerrna unicolor ) Preparation method of secondary seed culture of strain 87613:

[0067] ① Will Cerrna unicolor The 87613 strain was cultured on a PDA plate for 4 days, and 5 mycelium pieces (1 cm in diameter) from the nascent area were taken and inoculated in PDB seed medium (filling volume 50 mL / 100 mL), at 30 °C, 200 rpm Cultivate under the conditions for 2 days to obtain a primary seed culture;

[0068] ②Transfer the primary seed culture to PDB medium (100 mL / 250 mL) at a ratio of 8% (v / v), and culture it for another 2 days under the same conditions to obtain the secondary seed cul...

Embodiment 2

[0073] Embodiment 2: Obtaining of laccase gene sequence

[0074] ①Extraction of total RNA: Collect the mycelium fermented on the 2nd day in the enzyme-producing medium of Isshiki 87613 by suction filtration, immediately add liquid nitrogen to grind, and use the operation steps of the RNeasy Plant Mini Kit kit to extract total RNA. The extracted RNA was detected by 2% agarose gel electrophoresis, and the OD260 / 280 ratio was measured by NanoDrop 2000 to check the purity of RNA.

[0075] ② Transcriptome library construction and sequencing

[0076] After the total RNA samples passed the test, Beijing Novogene Co., Ltd. performed transcriptome sequencing without reference. mRNA is isolated using magnetic beads with Oligo(dT) to perform A-T base pairing with polyA of mRNA. Fragmentation buffer was added to randomly fragment the complete mRNA fragments to form small fragments of about 200 bp. Under the action of reverse transcriptase, random primers are used to reverse transcribe ...

Embodiment 3

[0086] Embodiment 3: LacB laccase protein purification

[0087] Determination of protein concentration: The protein concentration was determined using the BCA kit (Transgen). Draw a standard curve according to the method in the manual, and calculate the protein concentration of the sample to be tested according to the standard curve.

[0088]

[0089]

[0090]

[0091] ①Use AKTA equipment and anion exchange column to purify protein, solution A is 50 mmol / L Tris-HCl buffer solution with pH 8.5, solution B is 50 mmol / L Tris-HCl buffer solution with pH 8.5, containing 1 mol / L NaCl solution, and the anion exchange column was equilibrated with solution A before loading the sample. After the sample was loaded, the elution program was set to 10% solution B, and the flow rate was 1 mL / min. The eluate with enzyme activity was collected, and the high enzyme activity group was separated Store in a 4°C refrigerator for later use. After this step of purification, the purification...

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Abstract

The invention provides cerrena unicolor derived laccase and a gene and application thereof, and belongs to the field of biotechnology. The amino acid sequence of the cerrena unicolor derived LacB laccase provided by the invention is as shown in SEQID NO.3, the overall-length sequence of the gene of the cerrena unicolor derived LacB laccase is as shown in SEQID NO.1, and the cDNA sequence is as shown in SEQID NO.2. The invention further discloses a fermentation production method of the LacB laccase. The LacB laccase is produced through fermentation of the cerrena unicolor, after the sixth day of fermentation culture, the laccase yield can reach 652 U / mL, and the fermentation production method has the advantages of being high in yield and short in enzyme production cycle. The LacB laccase has significant effects in environmental hormone degradation, and the degradation rate on diethylstilbestrol, hexestrol, dienestrol, beta-estradiol, bisphenol a and hydroprednisone is 98% or above. TheLacB laccase provides a new way for environmental pollutant government, and has good economic benefits and social benefits.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a laccase derived from Dendrobium monochrome and its gene and application. Background technique [0002] Laccase (Laccase, EC1.10.3.2) is a class of copper-containing polyphenol oxidase, which belongs to the blue multi-copper oxidase (blue multi-copper oxidase) family, and widely exists in fungi, plants, and insects. Among them, the white-rot fungi of the Basidiomycota are the main laccase-producing bacteria, which have excellent ability to degrade cellulose and hemicellulose. Current research shows that laccase can efficiently and cleanly remove a variety of persistent organic pollutants (including dyes, various antibiotics and hormone-containing drugs, phenol, aromatic amines and their derivatives, etc.) that cannot be effectively removed by traditional physical and chemical methods. ), which has attracted extensive attention for the application of laccase in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N1/14A62D3/02C12R1/645A62D101/26
CPCA62D3/02A62D2101/26C12N1/14C12N9/0061C12Y110/03002
Inventor 杨捷林沚葳姚炎华叶秀云
Owner FUZHOU UNIV
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