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Laccase-producing Coriolopsis gallica NCULAC F1 and method for preparing high-activity laccase liquid

A high-activity, coarse-haired technology, applied in the field of microbial enzymology, can solve the problem of low enzyme activity, and achieve the effect of high extracellular laccase activity, good application value, and high tolerance

Pending Publication Date: 2021-11-05
NANCHANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of low enzyme activity in the production of laccase by fungal fermentation at present, to provide a high-yield laccase-producing white-rot fungus-Porina pilosa, and a method for preparing a high-activity laccase liquid by using it

Method used

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  • Laccase-producing Coriolopsis gallica NCULAC F1 and method for preparing high-activity laccase liquid
  • Laccase-producing Coriolopsis gallica NCULAC F1 and method for preparing high-activity laccase liquid
  • Laccase-producing Coriolopsis gallica NCULAC F1 and method for preparing high-activity laccase liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Laccase-producing strain screening and identification

[0020] The decayed wood samples were collected in Nanchang, Jiangxi. Weighed 10g of the decayed wood samples and placed them in 100mL of sterile water containing 5 glass beads, shaken on a shaker for 20min, and diluted 10mL of the sample solution to 10 -1 、10 -2 、10 -3 . Make 5 parallels for each dilution, respectively take 100 μL of the dilution to a PDA solid plate containing 0.04% (v / v) guaiacol, spread it evenly with a coating rod, and place the plate upside down in a 28°C incubator Culture 7d. Pick reddish-brown colonies for plate streaking until pure colonies are obtained.

[0021] According to the size and color depth of the color circle, three strains of laccase-producing fungi were screened out, among which the reddish-brown color around the colony of strain NCULAC F1 was the deepest. It was judged that the strain NCULAC F1 had a strong ability to produce laccase, and it was preserved on the ...

Embodiment 2

[0022] Example 2 Production of high-activity laccase by liquid fermentation of Inonotus roughae NCULAC F1

[0023] Prepare PDA liquid medium, add 100mL medium to each 250mL Erlenmeyer flask, add copper sulfate at a final concentration of 0.25-5.0mM, and investigate the effect of copper sulfate addition on the expression of extracellular laccase in Inonotus pilosa NCULAC F1.

[0024] Inoculate the mycelial suspension of Inonotus crescens NCULAC F1 into PDA liquid medium according to the inoculum amount of 5% (v / v), and culture it in shake flask at 30° C. and 120 rpm for 15 days. From the third day of cultivation, 1 mL of fermentation broth was taken every day, centrifuged at 4°C and 6000 rpm for 3 min, and the supernatant was taken to measure laccase activity.

[0025] Laccase activity was determined using ABTS as the catalytic substrate. The 3mL total reaction system consisted of 2mL citric acid-disodium hydrogen phosphate buffer (0.1M, pH 4.5), 0.9mL ABTS (1mM) and 0.1mL enzy...

Embodiment 3

[0029] Example 3 Effects of Temperature and pH on the Activity and Stability of Inonotus pilosa NCULAC F1 Laccase

[0030]1. Effects of temperature on the activity and stability of laccase from Inonotus pilosa NCULAC F1

[0031] Place the laccase solution at 30-80°C for 5 minutes to incubate the activity of the laccase of Inonotus roughans, set the maximum enzyme activity as 100%, and calculate the relative enzyme activities at these temperatures to determine The optimum reaction temperature of laccase. Three parallels were performed in each group, and the average value was taken. Place the laccase solution in water baths at different temperatures (40, 50, 60, 70°C) for 5 hours, measure the remaining laccase activity every 1 hour, set the initial enzyme activity as 100%, and explore the laccase activity at different temperatures. down stability.

[0032] The results of the influence of temperature on the activity of NCULAC F1 laccase from Inonotus roughae are as follows: ...

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Abstract

The invention discloses laccase-producing Coriolopsis gallica NCULAC F1. The laccase-producing Coriolopsis gallica is preserved in China Center for Type Culture Collection on June 17, 2021, the preservation number of the laccase-producing Coriolopsis gallica is CCTCC No.M2021731, and the preservation address of the laccase-producing Coriolopsis gallica is No.299, Bayi Road, Wuchang District, Wuhan, Hubei Province. The Coriolopsis gallica NCULAC F1 disclosed by the invention has the following characteristics that the laccase-producing activity is high, and produced laccase has good thermal stability and has relatively high tolerance to metal ions, organic solvents and the like. The Coriolopsis gallica NCULAC F1 is used as an enzyme-producing strain, and a proper amount of copper sulfate is added into a PDA liquid culture medium, so that the yield of the extracellular laccase with high activity expressed by the Coriolopsis gallica is greatly improved. The method for preparing a high-activity laccase crude solution from the Coriolopsis gallica NCULAC F1 by adding the copper sulfate has the characteristics of simplicity and convenience in operation, simple process, good repeatability and the like, and the prepared laccase solution is relatively high in activity and has good application potential and development and utilization prospects in the industrial field.

Description

technical field [0001] The invention relates to the field of microbial enzymology, in particular to a laccase-producing Inonotus indigo NCULAC F1 and a method for preparing a high-activity laccase solution. Background technique [0002] Laccase (laccase, EC 1.10.3.2, referred to as Lac) is a polyphenol oxidase with a wide range of substrates, and has good oxidation effects on phenols, aromatic amines and other substances. , Dye decolorization and food industry and other fields have been widely used. With the rapid development of biotechnology, the demand for highly active laccase in many industrial fields is increasing, and its production cost, output and production efficiency have become important factors affecting the development of the laccase industry. In particular, the yield of laccase is the premise of its industrial application, and the shortage of yield directly restricts its application level. [0003] The source of laccase is very extensive, including organisms ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/02C12R1/645
CPCC12N9/0061C12N1/14C12Y110/03002
Inventor 巫小丹岑庆静卢艳娟崔宪刘玉环徐立娜付桂明洪妮李绪万辉刘薇
Owner NANCHANG UNIV
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