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Liquid fermentation method for producing laccase by using marine endogenous pestalotia

A technology of liquid fermentation of Polychaeta pseudodiscoides, which is applied in the direction of fungi and oxidoreductases, can solve the problems of unsatisfactory stability and environmental tolerance, and the limitation of lignin degradation efficiency, and achieve extensive process and easy Effects of control and scale-up, medium simplicity

Inactive Publication Date: 2011-07-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The stability and environmental tolerance of laccase produced by terrestrial microorganisms are generally not particularly ideal, and the degradation of lignocellulosic plants generally needs to be carried out under rough conditions, and its lignin degradation efficiency is often limited. Therefore, it is necessary to screen strains with better performance to produce laccase with high enzyme activity, good stability and environmental tolerance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 1) Strain screening:

[0039] Sludge and seaweed samples were taken from the seabed at a depth of 10m in the East China Sea, 5g of the sample was added to 50ml of sterilized artificial seawater, shaken at 160r / m for 20min, left to stand for 10min, and 0.25ml of the supernatant was taken, coated on a PDA plate, at 26°C, Cultivate for 15 days; pick the colony, inoculate it on the isolation selective medium, and cultivate it at 26°C. If the laccase-producing strain is isolated, it will appear on the selective medium containing guaiacol due to the oxidation of guaiacol The color is reddish brown, the darker the color, and the larger the range, the stronger the ability of the strain to produce laccase. The laccase-producing strain was inoculated in a liquid enzyme-producing medium for fermentation and cultured at 26°C for 8 days to measure the laccase activity. The target strain with high enzyme activity is the target strain. According to the ITS analysis of the strain and t...

Embodiment 2

[0049] 1) Strain screening:

[0050] Sludge and seaweed samples were taken from the seabed at a depth of 20m in the East China Sea, 10g of the sample was added to 50ml of sterilized artificial seawater, shaken at a speed of 250r / m for 30min, left to stand for 20min, and 0.5ml of the supernatant was taken, coated on a PDA plate, at 30°C, Cultivate for 20 days; pick the colony, inoculate it on the isolation selective medium, and cultivate it at 30°C. If the laccase-producing strain is isolated, it will appear on the selective medium containing guaiacol due to the oxidation of guaiacol. The color is reddish brown, the darker the color, and the larger the range, the stronger the ability of the strain to produce laccase. The laccase-producing strain was inoculated in a liquid enzyme-producing medium for fermentation and cultured at 30°C for 12 days to measure the laccase activity. The target strain with high enzyme activity is the target strain. According to the ITS analysis of the...

Embodiment 3

[0060] The first-class seed liquid of endophytic discoid polychaetes is inoculated on the liquid fermentation medium with 5% (v / v) inoculum, liquid medium composition: glucose 10g, ammonium tartrate 2g, KH 2 PO 4 1g, Na 2 HPO 4 0.2g, CuSO 4 ·5H 2 O 200μM (final concentration), trace element solution 10ml, yeast extract: 2g, adjust the pH to 5.5, and dilute to 1000ml with tap water. At a temperature of 28° C., a liquid content of 40%, a fermentation initial pH of 5.5, and 8-10 days of fermentation, the liquid fermentation laccase activity reaches 421.5 IU / ml. Enzyme activity unit IU is defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute as one laccase activity unit. Enzyme activity assay method: the fermentation medium was centrifuged at 10000r / m for 10min, and the supernatant was the crude enzyme solution. Take 0.1ml of properly diluted crude enzyme solution, add 2.5ml of NaAc-HAc (pH4.5) buffer solution, mix well, add 0.4ml of ABTS, react at 25°...

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Abstract

The invention discloses a liquid fermentation method for producing laccase by using marine endogenous pestalotia, which comprises the following steps of: (1) screening a strain: screening soil in the depth of 10-20m on the offshore of the East China Sea to obtain a novel marine fungi; (2) preserving the strain: inoculating the strain in a modified potato dextrose agar (PDA) medium for culture, and preserving on a slope at the temperature of 4DEG C; (3) activating and propagating the strain: inoculating a spore of the strain in a PDA medium added with special components, and inoculating the activated spore in a seed culture medium for propagation; (4) preparing a marine endogenous pestalotia liquid fermentation medium; and (5) fermenting: performing liquid fermentation and enzyme production by using the newly prepared medium, and determining the optimized enzyme production condition. In the method for producing the laccase, the novel marine endogenous pestalotia is taken as a production strain; and the method has the characteristic that the media are coarse, wide in sources, low in prices and the like.

Description

technical field [0001] The invention relates to a liquid fermentation method for producing laccase by using a marine endophytic Discotrichum pseudochaete. Background technique [0002] Lignin is the second largest renewable resource in nature after cellulose, but because of its complex structure and very stable chemical properties, it is recognized as one of the refractory aromatic compounds, which has been unable to be effectively utilized for a long time. It has become an important waste in industrial production and a serious environmental pollutant. [0003] Due to the mosaic combination of lignin, hemicellulose, and cellulose, lignin wraps cellulose and hemicellulose with matrix, deposits or fills in the microcrystalline fibers composed of cellulose, forming a natural barrier, which seriously hinders the formation of fibers. Biodegradation and utilization of toxins. Therefore, the difficult degradation of lignin has become the biggest obstacle to the efficient conversi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/02
Inventor 姚善泾陈慧英冯晓雨薛栋升林东强
Owner ZHEJIANG UNIV
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