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
View PDF2 Cites 4 Cited by
  • 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 pr

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Example Embodiment

[0036] Example 1

[0037] (1) Acquisition of Coprinus comatus mycelium

[0038] The Coprinus comatus strains were inoculated on the PDA plate from the inclined surface of the test tube and placed in a constant temperature incubator at 25°C for culture. 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 place them in each sterilized 250 mL Erlenmeyer flask (each The bottle contains 30mL liquid culture medium), placed in a constant temperature incubator at 25°C for culture. On the 10th day, caffeic acid (final concentration of 1mM) was added for induction, and the hyphae were collected on the 22nd day. The mycelium was filtered with gauze, then washed twice with PBS, quickly frozen in liquid nitrogen, and stored in a refrigerator at -80°C for later use.

[0039] (2) Extraction of total RNA

[0040] Extraction with Invitrogen's TRIZOL reagent, the specific process: ...

Example Embodiment

[0076] Example 2

[0077] The strains, reagents, and culture medium used in this example 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 Pl oymerase (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), other reagents are conventional reagents.

[0079] LBA solid medium: add peptone, yeast extract, and NaCl like LB medium, add 25g agar per liter of medium, autoclave at 121℃ for 20min, when the medium is cooled to 55℃, add Amp to make the final concentration Reach 100μg / mL, then pour it into a petri dish to make a flat plate.

[0080] LBZ (1L): 10g Tryptone, 5g Yeast Extract, 5g NaCl, autocl...

Example Embodiment

[0156] Example 3 Decolorization of dyes by recombinant Coprinus comatus laccase

[0157] Preparation of enzyme solution: culture the cloned recombinant laccase Lac6+10AA in a triangular flask at 25℃, 500MCu 2+ 1% methanol was added every 24h to induce expression. After 10 days of culture, the enzyme solution was collected, 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 a 2mL decolorization system, the final concentration of dye is 50mg / L, 0.1mol / L NaAc-HAc buffer, 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, the absorbance value A is measured in the same...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Molar extinction coefficientaaaaaaaaaa
Molar extinction coefficientaaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/53C12N9/02
Inventor 丁少军顾春娟王燕
Owner NANJING FORESTRY UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap