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Screening and identification method of electricigen enzyme

A technology of microorganisms and microbial strains, applied in the field of microbial enzymes, can solve problems such as interference with dyeing reactions and poor dyeing effects

Inactive Publication Date: 2015-03-25
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on the decolorization of dyes by bacterial laccases has been reported: Held C and other spore laccases and immobilized laccases decolorize three commonly used textile dyes DBPV200, DFB and isatin (the final concentration is 50mg / L). The spores can decolorize the dye very effectively within 90 minutes, and it is found that the bleached cotton fiber is dyed with CI reaction orange 70 or CI reaction blue, laccase can obviously interfere with the dyeing reaction, and the dyeing effect is not good

Method used

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  • Screening and identification method of electricigen enzyme
  • Screening and identification method of electricigen enzyme
  • Screening and identification method of electricigen enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Culture of Proteus hauseri

[0036] 1) Solid culture

[0037] Composition of LB medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L and agar 15g / L, pH7.0, inoculate the isolated and screened Proteus mirabilis on solid medium Incubate at 30°C for 16h.

[0038] 2) Liquid culture

[0039] First, a single colony was picked from the LB agar plate with an inoculation loop and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB culture solution at 30°C and 150 rpm for 12 hours.

[0040] 3) Dry weight analysis

[0041] Pour the bacterial suspension cultured for 12 hours into a glass filter group equipped with a 0.22 μm filter membrane, weigh the dry filter membrane at 50°C before installation, connect the suction pipe to the suction filter, and cover the filter group with a petri dish to prevent dust Invasion, pump air continuously until the bacteria liquid is partially dehydrated, then add deionized water to the bacteria liquid to diss...

Embodiment 2

[0042] Embodiment 2: SDS-polyacrylamide gel electrophoresis analysis

[0043] Prepare 10% separating gel in a small beaker, carefully pour the gel solution into the mold to avoid bubbles, and polymerize at room temperature for 30 minutes. Then prepare a 5% stacking gel solution, mix well and add it on top of the prepared separating gel, immediately insert a comb into the stacking gel, place it at room temperature for 1 hour to polymerize, and pull out the comb to form a sample hole. Put the whole set of colloids into the electrophoresis tank, add electrophoresis buffer (0.025mol / L tris, 0.192mol / L glycine, 0.1% sodium dodecylsulfonate, pH 8.3), and mix 30μL samples with 10 μL of quadruple protein loading buffer was mixed evenly, heated at 100°C for 5 min, and carefully added to the sample well for electrophoresis analysis. Adjust the voltage to 120V, perform electrophoresis separation for about 1.5 hours, take out the film and stain it with 0.025% Coomassie blue for 1 hour, a...

Embodiment 3

[0044] Example 3: Identification of Protein Identity by Tandem Mass Spectrometry

[0045] 1) Trypsinization:

[0046] Decolorization: add 100μL 50mmol / L NH to the centrifuge tube 4 HCO 3 / Acetonitrile (1:1) test dye gel decolorization solution, keep warm at 37°C for 20 minutes, then remove the solution, and wash repeatedly until the strip becomes colorless and transparent.

[0047] Dry gel: pipette the NH in the centrifuge tube 4 HCO 3 After aspirating with acetonitrile, add 100 μL of 100% acetonitrile, remove the acetonitrile after 10 minutes, and then put it in a 37°C oven for 5-10 minutes to ensure that the glue is completely dry.

[0048] Enzymolysis: Add 5-10 μL of trypsin solution with a concentration of 12.5 mg / L, and place in a 4°C refrigerator for about 30 minutes to ensure that the colloidal particles can absorb the enzyme solution well. After taking it out, enzymolyze in an oven at 37°C overnight.

[0049] Peptide extraction: add 60 μL of 50% acetonitrile and 0...

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Abstract

The invention relates to a screening and identification method of an electricigen enzyme and relates to a microbial enzyme. The method comprises the following steps: getting a genome message by proteomic analysis, and designing a random primer to perform gene cloning so as to get two brand new proteins, wherein the two brand new proteins are determined as laccase and porin F of Proteus penneri ATCC (American type culture collection) 35198 by comparison, and the amino acid similarity of each is 87.8% and 84.4% respectively. The laccase of electrogenesis Proteus and the enzyme activity characteristic, as well as the laccase and a porin gene are reported; and studies find that the enzyme activity of 269.2U / g-DCW can be obtained by using 2.5mmol / L of copper sulfate to perform induction production on the laccase, and the enzyme activity is improved by 30.6 times in comparison with that without induction. The invention provides a screening and protein identification method of a novel enzyme of an electricigen, which is expected to be applied to easing the crisis of growing shortage of petrochemical fuel and reducing the emission of carbon dioxide.

Description

technical field [0001] The invention relates to a microbial enzyme, in particular to a screening and identification method for an electrogenic microbial enzyme. Background technique [0002] Among the many renewable energy sources, the application of microorganisms to produce renewable electricity from organic waste is called "microbial fuel cell (MFC)". Microbial fuel cells are helpful in environmental protection, ecological balance and energy crisis. Using microorganisms to treat sewage and generate electricity at the same time has set off a research boom in the field of environment and energy (BruceE.Logan.Simultaneous wastewater treatment and biological electricity generation[J] . Water Science & Technology, 2005, 52:31-37). However, microbial fuel cells have not yet been commercialized. The main factors are: the performance of the microorganism itself, the type of organic waste to be decomposed, the life and power of the fuel cell, and the mode of operation. In the pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12R1/37
Inventor 吴意珣陈博彦卢英华郑雪松池小琴
Owner XIAMEN UNIV
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