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Laccase gene Lac6 and expression protein and application thereof

A gene and laccase technology, applied in the laccase gene Lac6 and its expression protein and application fields, can solve the problem of low expression of laccase, achieve good economic benefits, social effects, and good industrial application prospects

Inactive Publication Date: 2014-04-09
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression level of various laccases is very low. The cloning and sequence analysis of laccase genes to make them highly express recombinant laccase proteins on heterologous hosts is an important means to solve their high expression and be applied. Therefore, the heterologous expression of laccase has been studied

Method used

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  • Laccase gene Lac6 and expression protein and application thereof
  • Laccase gene Lac6 and expression protein and application thereof
  • Laccase gene Lac6 and expression protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Acquisition of mycelium of Coprinus comatus

[0038] The Coprinus comatus strains were inoculated from the slant of the test tube onto the PDA plate, and placed in a constant temperature incubator at 25°C for cultivation. Use a sterilized hole puncher with a diameter of 1 cm to punch holes on the PDA plate covered with mycelium (coprinus comatus takes about ten days), pick four colonies and put them in each sterilized 250mL triangular flask (each bottle containing 30mL liquid medium), placed in a constant temperature incubator at 25°C. On day 10, caffeic acid (final concentration: 1 mM) was added for induction, and mycelia were collected on day 22. The mycelium was filtered with gauze, washed twice with PBS, frozen in liquid nitrogen and stored in a -80°C refrigerator for later use.

[0039] (2) Extraction of total RNA

[0040] Using Invitrogen’s TRIZOL reagent for extraction, the specific process: take the Coprinus comatus sample out of the -80°C refrigerator, g...

Embodiment 2

[0077] The strains, reagents and culture medium used in this embodiment are as follows:

[0078] Yeast KM71H (Mut s , Arg + ) and the expression vector pPiczαB (Invitrogen). PGEM-T Easy Vector system I (Promega), Plasmid Miniprep Kit (BIOMIGA), AxyPrep PCR Clean up Kit (AXYGEN), EasySelectTM Pichia Expression Kit (Invitrogen), rTaqDNA Ploymerase (TakaRa), Pfu DNA Ploymerase (Promega), T4DNA Ligase (Promega, In vitrogen), Xba I (promega), sacII (Takara), Sac I (Fermentas), Amp (ampicillin SIGMA), EtBr (ethidium bromide AMERSCO), dNTP (TakaRa, Promega), Seakem LE agar ose (BMA), the rest of the reagents are routinely used reagents.

[0079] LBA solid medium: In addition to adding peptone, yeast extract, and NaCl as LB medium, add 25g agar per liter of medium, sterilize under high temperature and high pressure at 121°C for 20min, add Amp when the medium is cooled to 55°C, and make the final concentration Reach 100μg / mL, and then pour into a Petri dish to make a plate.

[008...

Embodiment 3

[0156] Embodiment 3 recombinant Coprinus comatus laccase is to the decolorization of dyestuff

[0157] Preparation of enzyme solution: culture cloned recombinant laccase Lac6+10AA in a Erlenmeyer flask at 25°C, 500MCu 2+ The expression was induced by adding 1% methanol every 24 hours, and the enzyme solution was collected after 10 days of culture, filtered by suction, concentrated by ultrafiltration, and stored at -80°C for later use.

[0158] (1) Decolorization of dyes by recombinant Coprinus comatus laccase at different times

[0159] In the 2mL decolorization system, the final concentration of dye is 50mg / L, 0.1mol / L NaAc-HAc buffer solution, the amount of enzyme is 0.5IU / mL, the final concentration of HBT is 0 or 0.1g / L, pH4.5, temperature 40℃, respectively When the time is 1, 2, 3, 4, 12h, measure the absorbance value of each reaction solution at the maximum absorption wavelength of the corresponding dye (A 1 ), while using the inactivated enzyme solution as a control, ...

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Abstract

The invention discloses a laccase gene Lac6 and expression protein and application thereof. The DNA sequence of the laccase gene Lac6 is shown in SEQ ID NO.1. According to the laccase gene Lac6 and the expression protein and application thereof, shown by research on the decoloration of recombinant coprinus comatus laccase through 13 kinds of dyes, the decoloration action of recombinant laccase Lac6+10AA mainly occurs in the beginning 1-2 hours of reaction, the decoloration speed is retarded along with time, the decoloration effect of pure laccase is obviously better than that of a crude laccase solution, and the decoloration ratio can be increased after HBT is added into crude laccase or pure laccase; the Lac6+10AA has a relatively good decoloration effect on remazol brilliant blue and malachite green, and the decoloration ratio can reach over 90% after the mediator HBT is added. The laccase gene Lac6 has a very good industrial application prospect and can generate relatively good economic and social effects.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a laccase gene Lac6 and its expression protein and application. Background technique [0002] Laccase is a copper-containing polyphenol oxidase, which is homologous to ascorbate oxidase and mammalian ceruloplasmin, and belongs to the blue multi-copper oxidase family. Because laccase has special catalytic properties and a wide range of substrates It has potential application value in textile dye decolorization and pulp bleaching, polymer synthesis, environmental detection, food industry, detoxification and bioremediation. Therefore, the research on the heterologous expression of laccase and its expression regulation has become the current international It is a research hotspot in the interdisciplinary field of enzyme engineering and environmental science. [0003] There are more and more studies on edible fungus laccase, for example, there have been relevant reports o...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02
Inventor 丁少军顾春娟王燕
Owner NANJING FORESTRY UNIV
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