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Method for preparing pycnoporus samguineus GW fungal laccase

A technology of Microporus hemoglobinus and laccase, which is applied in the field of microbial enzymology, can solve the problems of low yield and low yield of laccase, and achieve the effect of huge application potential.

Inactive Publication Date: 2006-07-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the laccase yields of the existing known strains are relatively low. According to the retrieved literature, domestic yields are all low, while foreign yields are relatively low.

Method used

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  • Method for preparing pycnoporus samguineus GW fungal laccase

Examples

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Embodiment 1

[0025] In the present invention, the mensuration of laccase activity is to adopt international general method, with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (abbreviated in English ABTS) {2, 2'-azino-bis-[3-ethylthiazoline-6-sulphonate]} to determine. The calculation of enzyme activity is carried out according to the definition of enzyme activity. The definition of an enzyme activity unit U: Under certain conditions, the amount of enzyme that catalyzes 1 μM substrate per minute. :

[0026] According to the Lambert-Beer law A=ε·L·C,

[0027] Get C=A / ε·L.

[0028] Obtain U=C·V / T according to the definition of enzyme activity,

[0029] Then the formula for calculating enzyme activity is U=A·V / (ε·L·T).

[0030] In the above formula, where C represents the amount of change in the concentration of the substrate within T time (unit: μM), A represents the change in the absorbance value of the UV spectrophotometer, and V repre...

Embodiment 2

[0033]Pycnoporus sanguineus (L: Fr) Murr. (GW) was inoculated onto a solid seed medium: wort, 2% agar. Culture at 30°C for 6-8 days. Get the bacterial block (diameter 1cm, 10 blocks) on the edge of the colony, inoculate into 100ml fermenting medium sterilized by autoclaving in a 500ml Erlenmeyer flask: 1.5% maltose, 0.2% ammonium tartrate, KH 2 PO 4 0.133%, NaH 2 PO 4 12H 2 O 0.039%, MgSO 4 ·7H 2 O 0.05%, sodium succinate (CH 2 COONa) 2 ·6H 2 O) 0.118%, FeSO 4 ·7H 2 O 0.00315%, CaCl 2 2H 2 O 0.01%, MnSO 4 ·H 2 O 0.0035%, CH 3 COONa·3H 2 O 0.0408%, CoCl 2 ·6H 2 O 0.006%, ZnSO 4 ·7H 2 O 0.0028%, CuSO 4 ·5H 2 O 0.0168%, the above "%" percentage is the percentage of water-based solid weight to water volume, wheat bran 6g, Tween-80 1ml, VitaminB1 10μg, VitaminB 25μg, VitaminB 65μg, add water to 1L. 8 pounds of autoclave for 30 minutes, 200 rpm, 30° C., dark culture, add inducer 2,5-dimethylaniline (2,5-Dimethylaniline) 10 μM on the fourth day, and cultivate f...

Embodiment 3

[0035] The fermented liquid obtained in Example 2 was taken, centrifuged to remove the fermentation sediment, the supernatant was vacuum filtered, and the obtained filtrate was ultrafiltered through a PLGC membrane (10000D) for preliminary concentration. Then two-step ammonium sulfate precipitation method is adopted for precipitation, the first step is to stir and precipitate with 35% (W / V) saturated ammonium sulfate at 4°C, centrifuge at 10,000rpm for 1 hour, discard the precipitate, and leave the supernatant; the second step uses the obtained product from the first step Add 80% (W / V) saturated ammonium sulfate to the supernatant at 4°C and gently stir to precipitate, let it stand at 4°C for more than 2 hours, then centrifuge at 10,000rpm for 1 hour, discard the supernatant, and leave the precipitate. The precipitate obtained in the second step was dissolved with twice the volume of 100 mM potassium phosphate buffer (pH 5.7), and then dialyzed, and the dialysate was 10 mM pota...

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Abstract

The present invention discloses one kind of sanguineous Pycnoporus GW (CGMCC No. 1008) strain different from available laccase and its parent strains. The present invention also provides the method of fermentation culturing the strain to prepare laccase. The strain has secondary hyphae laccase producing capacity as high as 43 U / ml. The laccase oxidizing substrate includes phenol and its derivative, aromatic amine and its derivative, aromatic carboxylic acid and its derivative, etc. The present invention may take important role of replacing chemical adhesive in bleaching paper making pulp, waste water treatment and timber processing.

Description

technical field [0001] The invention belongs to the field of microbial enzymology, and specifically relates to a new bacterial strain of Pycnoporus sanguineus (L: Fr) Murr.) CGMCC NO.1008 of the fungus Basidiomycota and a method for preparing laccase by cultivating the bacterial strain method. 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. Laccase can degrade the phenolic and non-phenolic structures in lignin, and plays an important role in the biodegradation of lignin. The degradation of lignin by enzymes is oxidative and non-specific, which makes laccases also capable of degrading aromatic compounds. The substrates oxidized by laccase include: phenols, aromatic amines, aromatic carboxylic acids, etc. At present, through continuous research, the range of non-phenolic substrates that can be oxidized by laccase is still expandi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N1/14C07K1/36C02F3/00C12R1/645
Inventor 郭林王文惠张虎成李伟
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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