CN1262645C - Method for preparing pycnoporus samguineus GW fungal laccase - Google Patents

Abstract

The invention discloses a strain of dense poriobacterium hemorrhoids GW (CGMCC NO.1008) which is different from other known laccase-producing similar strains and their parent strains. It also provides a method for preparing laccase by fermenting and cultivating the bacteria. The dikaryotic hyphae of the strain can produce enzymes up to 43U/mL. The substrates oxidized by laccase include: phenols, aromatic amines, aromatic carboxylic acids, etc. For this reason, the significance of the present invention plays an important role in the papermaking pulp biological bleaching and wastewater treatment, and in the wood processing industry to replace chemical adhesives .

Description

A kind of preparation method of GW laccase

technical field

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

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 expanding. Laccase can directly reduce molecular oxygen to water and catalyze the oxidation of organic pollutants in the absence of H ₂ O ₂ . Laccase can also oxidize and polymerize lignin polyphenols in the form of free radicals, replacing the use of synthetic resin adhesives or other chemicals in the traditional wood industry. Use non-polluting, low-energy enzymatic methods to replace heavily polluting chemical methods, and eliminate pollution at the source. Therefore, laccase has great application potential in biotechnology and environmental protection.

White rot fungi are the only organisms that can completely degrade lignin into carbon dioxide and water. White rot fungi are also capable of producing lignin peroxidase (LiP) and manganese oxidase (MnP), but the laccases they produce are more thermostable than lignin peroxidase and manganese peroxidase. Therefore, this biological group has aroused great interest of world biologists.

Among the white rot fungi, Microporosa haemophilus is considered to be an ideal strain for studying laccase. However, the laccase yields of the existing known strains are relatively low. According to the retrieved literature, the domestic yields are all low, while the foreign strains have relatively high yields. Therefore, the research on high-yielding strains of laccase is imminent, and thus it is necessary to provide new strains capable of producing laccase with high yield and relatively easy.

Contents of the invention

The object of the present invention is to provide a new mutant strain of Pycnoporus sanguineus (L: Fr) Murr. which can produce laccase. Another purpose is to provide GW CGMCC NO.1008 mutated strains of Microporosa hemoglobinum to be cultivated first; then from these cultures, conventional enzyme separation techniques such as: centrifugal separation, ultrafiltration concentration, ammonium sulfate fractional precipitation, ion exchange column , gel filtration column and freeze-drying provide a method for extracting and preparing laccase. The mutant strain can produce a large amount of laccase with high activity. According to the method provided by the present invention, the highest laccase activity produced by the dikaryotic hyphae of GWCGMCC NO.1008 in the liquid medium can reach 43U/mL.

The new mutant strain of the present invention Pycnoporus sanguineus (L: Fr) Murr.) GW has been on September 23, 2003 in the General Microbiology Center of China Microbiological Culture Collection Management Committee, China, Beijing, preservation, and received To deposit registration number: Pycnoporus sanguineus (L: Fr) Murr.) GW CGMCC NO.1008.

The GW GWCGMCC NO.1008 of the present invention is obtained by mutating the parent Pycnophora G05 through ultraviolet irradiation.

Bacterial strain Microporosa hemorrhoids GW CGMCC NO.1008 of the present invention has the following microbiological characteristics:

1. Morphological characteristics

Under the microscope, the hyphae grown by the bacterial strain of the present invention on the solid medium are easy to break.

The difference in morphological characteristics between the parental strain (G05) and the bacterial strain of the present invention GW CGMCC NO.1008 is shown in Table 1.

Table 1 Morphological properties Microporosa haemorrhoids GW (the strain of the present invention) GO5 (wild type strain) Colony morphology The colonies are round and ring-shaped. The colony is round without ring pattern. Mycelial morphology The hyphae are easily broken. Mycelium is not easy to break.

2. Physiological characteristics

The bacterial strain of the present invention is a kind of aerobic bacterium, shows optimal growth ability at 30 ℃, and it produces laccase in fermentation medium, and a large amount of pigments are accompanied to produce, after adding wheat bran, the laccase Yield will increase 2-3 times. The growth rate of the strains was not much different when using maltose, sucrose or glucose as carbon source, but the amount of laccase produced was higher when using maltose than when using sucrose or glucose.

The method for preparing laccase by using the bacterial strain of the present invention includes the method of cultivating Microporosa haemophilus GW in a medium containing carbon source, nitrogen source, inorganic matter and other nutrients, and the solid seed medium is: wort juice, 2% agar ; Fermentation medium: maltose 1.5%, ammonium tartrate 0.2%, 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, VitaminB2 5μg, VitaminB6 5μg, add water to 1L. Also included are methods of recovering laccase from culture.

The primary carbon source used in the medium of the present invention may include, but is not limited to, maltose. The nitrogen source used in the cultivation process of the present invention may include but not limited to ammonium tartrate. The inorganic components used in the fermentation of the present invention may include ferrous sulfate, magnesium sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium chloride, cobalt chloride and the like.

On the fourth day, 10 μM inducer 2,5-dimethylaniline (2,5-Dimethylaniline) was added, and the fermentation could be carried out under aerobic conditions at 30±0.1° C. for 12 days. The laccase product produced by the strain is secreted extracellularly and stored in the culture medium from which the laccase is isolated by using conventional isolation methods, but not limited to these procedures. For example: remove hyphae by centrifugation, use ultrafiltration to remove a part of small molecular substances and further concentrate the fermentation supernatant, ammonium sulfate fractional precipitation, ion exchange column, gel filtration column, freeze-drying. Most of the foreign protein and almost all of the pigment can be removed by these steps.

It has been confirmed by the present inventors that under the present conditions, the laccase activity obtained by fermentation of the binucleate hyphae of the present invention can be as high as 43U/ml. Moreover, the bacterium of the present invention does not produce lignin peroxidase and manganese peroxidase, which facilitates the extraction of laccase.

The laccase prepared according to the method provided by the invention can be applied in the biodegradation of phenols, aromatic amines and aromatic carboxylic acids. In addition, it can be used in paper pulping biological bleaching and wastewater treatment and wood processing instead of chemical adhesives. Therefore, the significance of the present invention is that it can replace the heavily polluting chemical method synthetic resin adhesive or other chemicals used in the traditional wood processing industry, and adopt the pollution-free, low-energy laccase of the present invention to replace the traditional wood processing industry. It has great application potential in environmental protection.

Description of drawings

Figure 1: Colony morphology of GW CGMCC NO.1008

Figure 2: Mycelial morphology of GW CGMCC NO.1008 (x1100)

Detailed ways

The present invention will be further illustrated by the following examples, but the present invention is not limited by these

EXAMPLE LIMITATIONS.

Example 1

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. :

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

Get C＝A/ε·L.

  According to the definition of enzyme activity, U=C V/T is obtained,

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

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 represents the volume of the reaction system (unit: mL) , ε=3.6×10 ⁴ M·cm ^-1 ＝36(μmol/ml) ^-1 ·cm ^-1 , is the molar extinction coefficient of oxidized ABTS, L is the optical path of the cuvette (unit: cm), T is the reaction time (unit: minute).

The enzyme activity measurement conditions in the present invention are: reaction volume V=1mL, light path L=0.5cm of cuvette, reaction time T=30 seconds. The reaction system contains 500 μL of 50 mM tartrate buffer (pH4), 390 μL of water, 100 μL of 500 μM ABTS, 10 μL of fermentation broth in a volume of 1 mL, and the wavelength is 420 nm. If the laccase activity in the fermentation broth is too high, it can be diluted appropriately.

Example 2

Pycnoporus sanguineus (L: Fr) Murr. (GW) was inoculated onto a solid seed medium: wort, 2% agar. Culture at 30°C for 6-8 days. Take the bacterial block (diameter 1cm, 10 pieces) at the edge of the colony and inoculate it into a 500ml Erlenmeyer flask filled with 100ml fermentation medium sterilized by autoclaving: maltose 1.5%, ammonium tartrate 0.2%, 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 for 12 days. The laccase activity was measured according to the method in Example 1, and the laccase activity obtained by mycelia fermentation could be as high as 43 U/ml.

Example 3

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 potassium phosphate buffer. The yield of laccase after ultrafiltration is 81.82%, the yield of laccase after ammonium sulfate precipitation is 88.89%, and the yield of laccase after potassium phosphate buffer dialysis is 96.46%.

Example 4

In this example, the laccase protein was further isolated and purified. Take 10 mL of the concentrated solution in Example 3 for ion exchange column chromatography. The ion exchange medium is DEAE Sepharose F.F., the buffer is 20mM histidine buffer (pH6.0), the eluent is 0.1-0.6M NaCl histidine eluent, and the flow rate is 1mL/min. Then freeze-dry and concentrate to obtain laccase.

Claims (8)

1. A method for preparing laccase by fermenting Microporosa haemorrhoids GW CGMCC NO.1008, the method comprising: first cultivating Microporosa haemophilus GW CGMCC NO.1008; then from these cultures by centrifugation, ultrafiltration and concentration , ammonium sulfate fractional precipitation, ion exchange column, gel filtration column, and freeze-drying separation technology to extract and prepare laccase, wherein the first culture is carried out under the conditions of aerobic and dark culture. 2. The method according to claim 1, the carbon source used to cultivate earlier is maltose, the nitrogen source has ammonium tartrate, and the inorganic components used have ferrous sulfate, magnesium sulfate, manganese sulfate, zinc sulfate, copper sulfate, chloride Calcium and Cobalt Chloride. 3. _The method according to claim 1, the solid seed medium is: wort juice, 2% agar; the fermentation medium is: maltose 1.5%, ammonium tartrate 0.2%, KH ₂ PO 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 solid weight to water volume based on water, wheat bran 6g, Tween-801ml, vitamin B110μg, vitamin B25μg, vitamin B65μg, add water to 1L. 4. according to the described method of claim 1, dense porosity haemophilus GW CGMCC NO.1008 cultivated through 4 days, culture medium will add inducer 2,5-dimethylaniline, from the 12th day of beginning to cultivate, from cultivating Isolate and purify laccase from the material, and the enzyme activity is as high as 43U/mL. 5. The method according to claim 4, the amount of the inducer 2,5-dimethylaniline added to the culture medium is 10 μM. 6. the application of the laccase prepared by the method described in claims 1-5 in degrading phenols, aromatic amines and aromatic carboxylic acids. 7. The application of the laccase prepared by the method according to claims 1-5 in biological bleaching of paper pulping and wastewater treatment. 8. the application of the laccase prepared by the method described in claims 1-5 in wood processing instead of chemical adhesives.