Laccase and preparation method and special production strain thereof

A technology for laccase and derivatives, applied in the field of microbial enzymology, can solve problems such as low laccase activity, and achieve the effect of simple and convenient preparation method

Inactive Publication Date: 2010-11-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently known strains have low laccase activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, fermenting and cultivating Microporosa haemophilus MK2001 to prepare laccase

[0023] 1. Fermentation and cultivation of Microporosa haemophilus MK2001

[0024] Solid seed medium: wort medium plate containing 2g / 100ml agar.

[0025] Fermentation medium: maltose 15g, ammonium tartrate 2g, KH 2 PO 4 1.33g, NaH 2 PO 4 12H 2 O 0.39g, MgSO 4 ·7H 2 O 0.5g, sodium succinate (CH 2 COONa) 2 ·6H 2 O 1.18g, FeSO 4 ·7H 2 O 0.0315g, CaCl 2 2H 2 O0.1g, MnSO 4 ·H 2 O 0.035g, CH 3 COONa·3H 2 O 0.408g, CoCl 2 ·6H 2 O 0.06g, ZnSO 4 ·7H 2 O 0.028g, CuSO 4 ·5H 2 O 0.168g, wheat bran 7.5g, corn flour 25g, Tween-80 1ml, Vitamin B 1 10 μg, Vitamin B 2 5 μg, Vitamin B 6 5 μg, add water to 1L, and then sterilize with 8 pounds of high-pressure damp heat for 30 minutes.

[0026] Inoculate MK2001 into solid seed medium and culture at 30°C for 6-8 days. Take the most edge bacterial block (diameter 1cm, 10 blocks) of the colony, inoculate it on the 100ml...

Embodiment 2

[0036] Example 2. Pretreatment experiment of lignin degradation on rice straw by strain MK2001

[0037] The 250ml Erlenmeyer flask enzymatic hydrolysis system includes: laccase 1800U, rice straw 0.4g, 0.1M tartaric acid buffer 15ml (pH4.0), supplemented with sterile water to 20mL. The pretreatment temperature was 30° C., the rotating speed of the shaker was 150 rpm / min, and the treatment time was 24 hours, 48 ​​hours, and 72 hours, respectively. The method of lignin determination is Fan's method, and the experiment is repeated 3 times. The treatment results: 33.9% of lignin is degraded in 24 hours, 54.5% of lignin is degraded in 48 hours, and 56.3% of lignin is degraded in 72 hours.

[0038] In this example, a lignin degradation pretreatment experiment was carried out on rice straw, and the experimental results showed that: MK2001 laccase can effectively degrade lignin components in rice straw. The laccase produced by the strain MK2001 is used for the biological pretreatment ...

Embodiment 3

[0039] Example 3. Degradation treatment experiment of bacterial strain MK2001 on anthracene and benzo[a]pyrene in polycyclic aromatic hydrocarbons

[0040] Get each 4.5ml of enzyme solution respectively and place in 15ml brown reagent bottle with stopper, add the acetonitrile solution of 0.5ml anthracene and benzo[a]pyrene, make the acetonitrile content of reaction system reach 10% (percentage by volume), tighten the bottle stopper, Shake well, place in a dark room and incubate for 24 hours (28° C.), and then add 5 ml of acetonitrile to terminate the reaction. At this time, the content of acetonitrile is 55% (volume percentage). After shaking for 1 hour (28° C., 15000 rpm), centrifuge at high speed, pass through a 0.22 μm filter membrane, and perform sample determination by HPLC. For the control, pure water was used instead of the enzyme solution, and the other steps were the same. At the same time, laccase derived from Trametesversicolor (laccase (Tv)) was used as a set of t...

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Abstract

The invention discloses a laccase and a production method and a special production strain thereof and relates to the industrial field of microorganism fermentation. The strain provided by the invention is pycnoporus sanguineus MK 2001 CGMCC No.2932. The laccase provided by the invention is prepared by fermenting the pycnoporus sanguineus MK 2001 CGMCC No.2932. The enzyme activity of fermented liquid laccase prepared by fermenting the pycnoporus sanguineus MK 2001 CGMCC No.2932 provided by the invention is up to 300 U/ml, and the laccase can be widely applied to biodegradation of phenols, arylamine, aromatic carboxylic acid and the derivatives of the phenols, the arylamine, and the aromatic carboxylic acid, enzyme bleaching of paper making and pulping, waste water treatment and dye degradation and can substitute a chemical adhesive in the wood processing.

Description

technical field [0001] The invention relates to the field of microbial enzymology, in particular to a laccase, a preparation method thereof and a special production strain. Background technique [0002] Laccase (p-diphenol oxygen oxidoreductase, EC 1.10.3.2.) is a blue phenol oxidase containing multiple copper ions, which can catalyze the redox reaction of phenols. The substrates oxidized by laccase include: phenols and their derivatives, aromatic amines and their derivatives, aromatic carboxylic acids and their derivatives, etc. At present, through continuous research, the range of non-phenolic substrates that can be oxidized by laccase is still expanding. Laccase (1accase) can degrade phenolic and non-phenolic structures in lignin, and plays an important role in the biodegradation of lignin; in addition, because many chemical bonds in lignin generally exist in aromatic compounds, and The degradation of lignin by laccase is oxidative and non-specific, which makes laccase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/02A62D3/02D21C9/10C02F3/34C02F1/72B09C1/10C02F103/30C02F103/28A62D101/28A62D101/26C12R1/645
Inventor 郭林李伟
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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