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Culture preparation method and application of trametes hirsuta laccase with dyestuff synergistic degradation effect

A technology of synergistic degradation and trachephrine bacteria, applied in the field of microbial enzymology, can solve the problems of no reports on the synergistic decolorization ability of laccase protein and less research on the induction and cultivation of laccase isozymes

Active Publication Date: 2017-03-15
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few studies on the induction and cultivation of laccase isozymes in the same fungus, especially the synergistic decolorization ability of different laccase proteins in the same fungal strain has not been reported

Method used

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  • Culture preparation method and application of trametes hirsuta laccase with dyestuff synergistic degradation effect
  • Culture preparation method and application of trametes hirsuta laccase with dyestuff synergistic degradation effect
  • Culture preparation method and application of trametes hirsuta laccase with dyestuff synergistic degradation effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, the induction culture of trametes hirsuta MX2 laccase

[0071] Inoculate the Trametes hirsuta MX2 preserved on the potato dextrose (PDA) slant medium to activate on the solid medium, cultivate it in a constant temperature incubator at 28°C for 7 days, and then take a 1*1cm square bacterial cake to the content of 100mL In a 250mL Erlenmeyer flask with fermentation medium, shake culture at a constant temperature of 150r / min and 28°C, take 1mL of the culture solution into a centrifuge tube every other day, centrifuge at 10000r / min at 4°C for 5min, and obtain the supernatant as a crude Enzyme liquid assay for laccase activity.

[0072] Laccase activity was determined with ABTS as substrate. Activity determination in 3mL reaction system: 2.8mL concentration of 0.5mmol / L ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) 0.05mol / L Sodium acetate buffer (pH 4.8), add 200 μL crude enzyme solution. Start the reaction at 25°C, measure the...

Embodiment 2-1

[0077] Example 2-1, Separation and Purification of Trametes hirsuta MX2 Laccase ThLac1 and ThLac2

[0078] Poplar sawdust was used as the carbon source of the fermentation medium, and the culture was shaken at a constant temperature of 150r / min and 28°C for 13 days. The fermentation broth was centrifuged at 4000rpm / min for 30min, and the supernatant after centrifugation was the crude enzyme solution. The crude enzyme solution is separated and purified through ultrafiltration concentration, ammonium sulfate precipitation dialysis, ion exchange chromatography and gel chromatography. The specific process is as follows:

[0079] ①, ultrafiltration concentration:

[0080] Put the crude enzyme solution into an ultrafiltration tube with a filter membrane pore size of 10 kDa, and centrifuge at 4°C and 4000 rpm / min for 30 minutes to obtain the ultrafiltrate.

[0081] ②, ammonium sulfate precipitation dialysis:

[0082] Slowly add ammonium sulfate to the ultrafiltrate so that the fina...

Embodiment 3

[0107] Embodiment 3, laccase ThLac1 and ThLac2 molecular weight determination and zymogram analysis

[0108] Molecular weight determination of purified laccases ThLac1 and ThLac2 was carried out by sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% resolving gel (w / v) and 5% stacking gel (w / v) estimate. After gel electrophoresis, it was stained with Coomassie Brilliant Blue R-250 and displayed and photographed for preservation, and then the molecular weight was estimated according to the standard protein marker in the gel.

[0109] The zymogram analysis of purified laccases ThLac1 and ThLac2 was carried out by non-denaturing polyacrylamide gel electrophoresis (native-PAGE) with 10% separating gel (w / v) and 5% stacking gel (w / v), wherein, the gel No SDS was added in the preparation, and the enzyme solution was directly loaded for electrophoresis without heat denaturation. After electrophoresis, the gel was taken out and placed in a staining solution...

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Abstract

The invention discloses a culture preparation method of trametes hirsuta laccase with a dyestuff synergistic degradation effect. The culture preparation method comprises the following steps: putting activated trametes hirsuta MX2 into a fermentation culture medium and culturing; centrifuging obtained fermentation liquid, wherein supernatant obtained by centrifuging is crude enzyme liquid; and separating and purifying the crude enzyme liquid to obtain two types of laccase isozymes which are marked as ThLac1 and ThLac2. The invention further provides a dyestuff de-coloring method which is carried out by utilizing the trametes hirsuta laccase; and the dyestuff de-coloring method comprises the following steps: mixing the ThLac1 with the ThLac2, wherein the ThLac1 accounts for 50%-80% of the total enzyme activity of a mixed enzyme; and putting the mixed enzyme into a dyestuff until the content is 50IU / L and de-coloring, wherein the de-coloring time is more than or equal to 3 hours.

Description

technical field [0001] The invention relates to the field of microbial enzymology, in particular to the cultivation and preparation method of a trachemetes laccase with synergistic effect on dye degradation. Background technique [0002] Laccase (laccase, EC 1.10.3.2) is a copper-containing polyphenol oxidase, which belongs to the cerulus oxidase family together with ascorbate oxidase in plants and ceruloplasmin in mammalian plasma. Laccase can obtain electrons from substrates or mediators, then transfer electrons to molecular oxygen, and finally form water. Due to the particularity of the catalytic mechanism of laccase, it acts on a wide range of substrates. According to statistics, laccase can catalyze the oxidation of more than 250 compounds in 6 categories, including phenols and their derivatives, aromatic amines and their derivatives, carboxylic acids and their derivatives, steroid hormones and biochromes, metal organic compounds, and Other non-phenolic substances, et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C02F3/34C12R1/645
CPCC02F3/342C12N9/0061C12Y110/03002
Inventor 潘程远朱兰兰王晨之
Owner ZHEJIANG FORESTRY UNIVERSITY
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