Effective method for rapidly and accurately screening lactase fungi

Abstract

The invention discloses an effective method for rapidly and accurately screening lactase fungi. The method sequentially includes the steps: 1) matching PDA culture media in a culture vessel, adding an appropriate amount of ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), and enabling the final concentration of the ABTS to range from 0.01% to 0.03%; 2) cutting plant materials (such as dead branches, fallen leaves, barks, crop straws and plant roots) into suitable small segments, placing the segments into the culture media, and performing culture for 1-48 hours at the constant temperature of 37 DEG C; 3) showing that the lactase fungi exist at culture media when the culture media on the periphery of small segments of plant organs appear green or purple reaction; 4) screening fungus hyphae from the culture media with color reaction, and continuing culture to achieve purification to obtain the lactase fungi.

Description

technical field

[0001] The invention relates to a screening method for laccase-producing fungi, in particular to an effective method for quickly screening laccase-producing fungi from the environment. Background technique

[0002] Laccase is a copper-containing polyphenol oxidase that is ubiquitous in fungi. Laccases are widely used in pulp bleaching,

[0003] Industrial and biotechnology fields such as dye decolorization, organic synthesis, and lignin degradation have very important economic and social values. Since different application fields require laccases with different enzymatic properties, it is of great significance to obtain laccases with diversity by developing new laccase gene resources. The gene diversity of laccase-producing fungi provides unlimited possibilities for extensive screening of laccase-producing fungi. The traditional screening of laccase-producing fungi is carried out by the guaiacol method, which uses the discoloration reaction between guaiaco...

Examples

Embodiment 1

[0011] Embodiment 1: first configure the PDA medium that contains 0.01% ABTS, then cut the small fragments of the dead branches that are collected into about 2cm, select 6 and place them on the prepared medium, and find that 5 dead branches are small after 1 hour. A purple reaction appears in the medium next to the fragment. After 12 hours of constant temperature cultivation at 37 degrees Celsius, the mycelia were cut and purified, and identified by molecular biology methods, all belonged to laccase-producing fungi.

Embodiment 2

[0012] Embodiment 2: configuration contains the PDA medium of 0.015% ABTS, then the bark of collection is cut into the fritter of about 1.5cm in length and width, selects 4 pieces and is placed on the prepared medium, after 18 hours of constant temperature cultivation at 37 degrees Celsius, it is found that 4 A green reaction occurs in the medium next to the bark nubs. The cut mycelia were purified and cultivated, and identified by molecular biological methods, all belonged to laccase-producing fungi.

Embodiment 3

[0013] Example 3: Configure the PDA medium containing 0.028% ABTS, then cut the collected rice straw into small fragments about 3 cm in length, select 8 pieces and place them on the prepared medium, and after cultivating at a constant temperature of 37 degrees Celsius for 35 hours, 3 of them were found The culture medium next to the rice straw has a purple reaction. The cut mycelia were purified and cultivated, and identified by molecular biological methods, all belonged to laccase-producing fungi.

[0014] Implementation effect: Using the above-mentioned scheme to screen laccase-producing fungi, there is no need to perform complicated pretreatment on the material in advance, just put the shredded plant material on the medium containing ABTS, once a green or purple reaction appears, it can be identified 100% The fungus is a laccase-producing fungus. Therefore, the sensitivity and efficiency of screening are greatly improved.

technical field

[0001] The invention relates to a screening method for laccase-producing fungi, in particular to an effective method for quickly screening laccase-producing fungi from the environment. Background technique

[0002] Laccase is a copper-containing polyphenol oxidase that is ubiquitous in fungi. Laccases are widely used in pulp bleaching,

[0003] Industrial and biotechnology fields such as dye decolorization, organic synthesis, and lignin degradation have very important economic and social values. Since different application fields require laccases with different enzymatic properties, it is of great significance to obtain laccases with diversity by developing new laccase gene resources. The gene diversity of laccase-producing fungi provides unlimited possibilities for extensive screening of laccase-producing fungi. The traditional screening of laccase-producing fungi is carried out by the guaiacol method, which uses the discoloration reaction between guaiaco...

Examples

Embodiment 1

[0011] Embodiment 1: first configure the PDA medium that contains 0.01% ABTS, then cut the small fragments of the dead branches that are collected into about 2cm, select 6 and place them on the prepared medium, and find that 5 dead branches are small after 1 hour. A purple reaction appears in the medium next to the fragment. After 12 hours of constant temperature cultivation at 37 degrees Celsius, the mycelia were cut and purified, and identified by molecular biology methods, all belonged to laccase-producing fungi.

Embodiment 2

[0012] Embodiment 2: configuration contains the PDA medium of 0.015% ABTS, then the bark of collection is cut into the fritter of about 1.5cm in length and width, selects 4 pieces and is placed on the prepared medium, after 18 hours of constant temperature cultivation at 37 degrees Celsius, it is found that 4 A green reaction occurs in the medium next to the bark nubs. The cut mycelia were purified and cultivated, and identified by molecular biological methods, all belonged to laccase-producing fungi.

Embodiment 3

[0013] Example 3: Configure the PDA medium containing 0.028% ABTS, then cut the collected rice straw into small fragments about 3 cm in length, select 8 pieces and place them on the prepared medium, and after cultivating at a constant temperature of 37 degrees Celsius for 35 hours, 3 of them were found The culture medium next to the rice straw has a purple reaction. The cut mycelia were purified and cultivated, and identified by molecular biological methods, all belonged to laccase-producing fungi.

[0014] Implementation effect: Using the above-mentioned scheme to screen laccase-producing fungi, there is no need to perform complicated pretreatment on the material in advance, just put the shredded plant material on the medium containing ABTS, once a green or purple reaction appears, it can be identified 100% The fungus is a laccase-producing fungus. Therefore, the sensitivity and efficiency of screening are greatly improved.

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