Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene

A technology for papermaking wastewater and laccase, applied in the biological field, can solve problems such as inability to completely eliminate wastewater, and achieve the effect of improving expression vitality

Inactive Publication Date: 2013-08-07
ZHEJIANG SHUANGLIANG SUNDA ENVIRONMENTAL PROTECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]Due to the lack of methods and culture substrates to reproduce environmental conditions, most microorganisms are difficult to recover and culture by standard laboratory methods, called uncultured microorganisms (uncultured) microorganism)

Method used

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  • Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene
  • Laccase for biological treatment of papermaking wastewater and encoding gene as well as expression and application of encoding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 : Construction of activated sludge DNA library

[0023] 1. Extraction of activated sludge DNA

[0024] Sludge was collected from a papermaking wastewater treatment plant, and was properly diluted as a sample for DNA extraction. 12000 rpm -1 Collect samples by centrifugation for 5 min, resuspend in TE and wash once, then add lysozyme to a final concentration of 100 μg·ml -1 , 37 ℃ water bath for 2 hours. Add 5 M NaCl to adjust the final concentration to 0.5 M, mix well, add 40 μl proteinase K and 20% SDS (final concentration is 0.5-1%), and store at 37 °C overnight or in a water bath at 50 °C for 3 h until the solution is viscous and clear. Add 1 / 3 volume of saturated NaCl, shake vigorously for 15 s, 12000 r·min -1 Centrifuge for 5 min, collect the supernatant into a sterilized centrifuge tube, add 0.6 V isopropanol to mix and precipitate, 12000 r min -1 The precipitate was collected by centrifugation, washed with 70% ethanol, dried and dissolved in 40...

Embodiment 2

[0034] Example 2 : Screening and sequence analysis of laccase genes

[0035] The clones in the DNA library were cultured and preserved separately, and the positive clones and the highly efficient laccase-producing transformants were screened.

[0036] Primary Screening Medium

[0037]Bavendamm's reaction medium: add tannic acid to the PDA solid medium to make the final concentration 0.4 mmol l / L;

[0038] Oxidation zone assay medium: Add guaiacol to PDA solid medium to make the final concentration 0.04%.

[0039] Re-screening medium

[0040] 200 g of potatoes, 20 g of glucose, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate heptahydrate, 1 g of yeast extract, 1 000 mL of distilled water, boil and filter, after cooling, divide into 250 mL Erlenmeyer flasks, each bottle Inject 50 mL of culture medium.

[0041] Enzyme-producing basal medium

[0042] Glucose 30 g, sodium nitrate 2 g, magnesium sulfate heptahydrate 0.5 g, dipotassium hydrogen phosphate tri...

Embodiment 3

[0050] Example 3 : Expression of laccase gene in yeast cells

[0051] 1. Construction of expression vector

[0052] Digest puc-LacN with EcoRI / NotⅠ, recover the exogenous fragment, insert it into the Pichia pastoris constitutive expression vector pGAPZaA (Invitrogen, USA) with the correct reading frame, connect and transform E. coli DH5α, the recombinant plasmid pGAPZaA-LacN was obtained. The recombinant plasmid was verified by EcoRI / NotI double enzyme digestion.

[0053] 2. Preparation of competent cells for electroporation

[0054] In a 50 ml centrifuge tube containing 5 ml YPD, culture yeast cells for 30 overnight; take 30~50 ul of the overnight culture, inoculate a 250 ml shake flask containing 50 ml of fresh medium, grow overnight until OD600=1.3~1.5 ;Collect the cells by centrifugation at 1500 g at 4°C for 5 min, and suspend the cells with 50 ml of pre-cooled sterile water; centrifuge as above, and suspend the cells with 25 ml of pre-cooled sterile water; Suspen...

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Abstract

The invention relates to the field of biotechnology, and discloses a laccase for biological treatment of papermaking wastewater as well as an encoding gene and application of the encoding gene in the biological treatment of the papermaking wastewater. The nucleotide sequence of the laccase gene is shown in SEQ IDNO:2, and the amino acid sequence of the laccase which is contains the gene codes is shown in SEQ IDNO:1. According to the laccase and the encoding gene, an efficient expression can be realized in microzyme, and the defects of the conventional pure culture technology and induced expression are overcome; and the laccase gene can improve the expression activity of the laccase, and is of great importance for promoting the application of the laccase in the biological treatment of the papermaking wastewater.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a laccase used for the biological treatment of papermaking wastewater, a coding gene and its application in the biological treatment of papermaking wastewater. Background technique [0002] my country's paper industry has a large amount of wastewater discharged annually, and the treatment is difficult. In addition to the promulgation and implementation of the "Discharge Standards for Water Pollutants in the Pulp and Paper Industry" (GB3544-2008), the treatment of paper industry wastewater has become more difficult and the treatment costs have increased significantly. With the enhancement of people's awareness of environmental protection, the continuous improvement of laws and regulations and the continuous strictness of the discharge indicators of papermaking wastewater, the treatment and compliance of papermaking wastewater have important practical significance. [0003] At ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/15C12N1/19C12N1/21C02F3/00C02F103/28
Inventor 郑展望杨瑾徐甦
Owner ZHEJIANG SHUANGLIANG SUNDA ENVIRONMENTAL PROTECTION CO LTD
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