CN1308435C - Laccase, preparing method thereof and strain special for same - Google Patents

Abstract

The invention discloses a laccase, its production method and a special production strain, and relates to the field of microbial fermentation industry. The bacterial strain provided by the present invention is Pycnoporus sanguineus mk528 CGMCC №1124. The laccase provided by the present invention is obtained by fermenting Pycnoporus sanguineus mk528 CGMCC №1124. The method for producing laccase comprises fermenting and culturing Pycnoporus sanguineus mk528 CGMCC No. 1124, and extracting laccase from the fermentation medium. Pycnoporus sanguineus mk528 CGMCC №1124 provided by the present invention is fermented and cultivated, and the laccase enzyme activity of the obtained fermentation liquid is as high as 63U/ml; and the bacterial strain does not produce lignin peroxidase and The manganese peroxidase is convenient for the extraction of laccase, and the extracted laccase can be widely used in papermaking wastewater treatment and in wood processing industry to replace formaldehyde and other chemicals.

Description

A kind of laccase and its production method and special production strain

technical field

The invention relates to the field of microbial fermentation industry, in particular to a method for producing laccase and a special bacterial strain thereof.

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 phenolic substances. Its reaction substrates include: phenolic and Its 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 can degrade phenolic and non-phenolic structures in lignin, which plays an important role in the biodegradation of lignin, and can also be used to degrade aromatic compounds. Laccase can also oxidatively polymerize lignin polyphenols in a free radical manner, which can replace synthetic resin adhesives or other chemicals used in the traditional wood industry, and replace heavily polluting chemical methods with this non-polluting enzymatic method , can eliminate pollution at the source. Therefore, laccase has great application potential in biotechnology and environmental protection.

White rot fungi can produce laccase, lignin peroxidase (LiP) and manganese oxidase (MnP), etc., and are currently the only organisms that can completely degrade lignin into carbon dioxide and water. The laccase produced by it is more thermostable than lignin peroxidase and manganese peroxidase. Therefore, this biological group has aroused great interest of world biologists. Among the white rot fungi, D. sanguineus is considered to be an ideal strain for studying laccase. However, the laccase enzyme activity of the known strains in China is relatively low, reaching up to 21U/ml.

Contents of the invention

The object of the present invention is to provide a laccase-producing Pycnophora haemorrhoids.

Pycnoporus sanguineus mk528 provided by the present invention has been preserved in the General Microorganism Center (CGMCC) of China Microbiological Culture Collection Management Committee (abbreviated as CGMCC) on March 25, 2004, and the preservation number is CGMCC № 1124.

The Pycnoporus sanguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124 was isolated from Pycnoporus sanguineus G5. Its growth status on solid medium is shown in Figure 1, and the Giemsa staining of mycelial cells is shown in Figure 2 . The morphological differences between this strain and its parent strain G5 are shown in Table 1.

Table 1 Morphological differences of hyphae Morphological properties Microporosa haemorrhoids G5 (parental strain) MK528 (the strain of the present invention) Mycelial morphology lock joint no lock joint Hyphal nucleus Dual-core single core

Pycnoporus sanguineus mk528 CGMCC № 1124 provided by the present invention is an aerobic bacterium with an optimum growth temperature of 30°C, and secretes laccase into the culture medium during fermentation, accompanied by a large amount of pigment produce.

Another object of the present invention is to provide a laccase and a method for producing laccase.

The laccase provided by the present invention is obtained by fermenting Pycnoporus sanguineus mk528 CGMCC No. 1124.

The method for producing laccase comprises fermenting and culturing Pycnoporus sanguineus mk528CGMCC № 1124, and extracting laccase from the fermentation medium.

The fermentation culture process needs to be carried out under aerobic and light-shielding conditions, and the inducer 2,5-dimethylaniline needs to be added to the culture medium until the fourth day, and the amount of 2,5-dimethylaniline added is preferably 10 μM. Used fermentation medium can use maltose, sucrose or glucose etc. as carbon source, has little influence on the growth rate of mycelia, but when maltose is used, laccase yield will be high; Culture medium usually uses ammonium tartrate as nitrogen source, also includes A variety of inorganic components, such as ferrous sulfate, magnesium sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium chloride, cobalt chloride, etc., and some nutrients, such as vitamin _B1 , _B2 , _B6 , etc. Adding wheat bran to the medium can increase the production of laccase by 2-3 times.

In practical application, the fermentation medium is preferably composed of the following substances, maltose: 1.0-2.0%; ammonium tartrate: 0.1-0.3%, KH ₂ PO ₄ : 0.132-0.134%; NaH ₂ PO ₄ ·12H ₂ O: 0.038-0.040%; MgSO ₄ 7H ₂ O: 0.04-0.06%; Sodium succinate (CH ₂ COONa) ₂ 6H ₂ O: 0.117-0.119%; FeSO ₄ 7H ₂ O: 0.00314-0.00316%; CaCl ₂ 2H ₂ O: 0.009-0.011%; MnSO ₄ H ₂ O: 0.0034-0.0036%; CH ₃ COONa 3H ₂ O: 0.0407-0.0409%; CoCl ₂ 6H ₂ O: 0.005-0.007%; ZnSO ₄ 7H ₂ O: 0.0027-0.0029%; CuSO ₄ 5H ₂ O: 0.0167-0.0169%; Wheat Bran 5-7g; Tween-80: 0.9-1.1ml; VitaminB ₁ : 10μg; VitaminB ₂ : 5μg; VitaminB ₆ : 5 μg, add water to 1 liter, wherein, said percentage is the percentage of water-based solid weight to water volume.

The extraction of laccase from the fermentation medium comprises the following steps: centrifugal separation, ultrafiltration concentration, ammonium sulfate fractional precipitation, ion exchange column and gel filtration column.

The fermented liquid is separated by centrifugation, the obtained supernatant is ultrafiltered with an ultrafiltration column with a molecular weight cut-off of 10,000 Daltons, and the concentrated solution is ultrafiltered with two-step ammonium sulfate precipitation, and the ammonium sulfate saturation of the two-step ammonium sulfate precipitation is respectively 30 %-40% and 70%-90%.

Pycnoporus sanguineus (Pycnoporus sanguineus) mk528 CGMCC № 1124 provided by the present invention is fermented and cultivated, and the laccase enzyme activity obtained can be as high as 63U/ml, and the bacterial strain does not produce lignin peroxidase and manganese during the culture process Peroxidase, which is convenient for the extraction of laccase, simplifies the extraction process, and the extracted laccase has high enzyme activity, which can be widely used in the biodegradation of phenols, aromatic amines, aromatic carboxylic acids and their derivatives, and the biodegradation of paper and pulp in bleaching and wastewater treatment, and can also replace chemical glues in wood processing.

Description of drawings

Figure 1 is a photo of the colony morphology of Microporosa haemorrhoids mk528 CGMCC № 1124

Fig. 2 is the Giema staining (×1100) micrograph of Microporosa haemorrhoids mk528 CGMCC № 1124

Detailed ways

Embodiment 1, the isolation of Microporosa hemorrhoids mk528

P. haemoporus mk528 was isolated from the dikaryotic hyphae of P. haemopore G5, while G5 was a dikaryotic hyphae obtained from the fruiting bodies of P. haemorrhoids (Tang Shun, Lomascolo A, Guo Lin, etc. (2001) Isolation of white-rot fungi from China for screening laccase-hyperproducing strains. Mycosystem 20: 520-525).

G5 was cultured in a petri dish containing agar 20g/l ^-1 and malt extract 20g/l ^-1 at 30°C. After 7 days of culture, the culture dish was inverted and kept at room temperature for 3-4 weeks . Then use high-temperature sterilized distilled water to collect basidiospores from the lid of the petri dish, and then dilute these basidiospore suspensions by an appropriate multiple, and apply them to the culture medium containing agar 20g/l ^-1 and malt extract 20g/l ^-1 On the second or third day, the visible basidiospore colonies were separated from the single spores in the culture dish, and the laccase high activity strain was screened by the method of syringaldazine, and the blood red dense pore was obtained. bacteria mk528.

Embodiment 2, fermenting and cultivating Microporosa haemorrhoids mk528 produces laccase

1. Fermentation of Microporosa haemophilus mk528

Solid seed medium: 2% malt extract, 2% agar.

Fermentation medium: maltose 15g, ammonium tartrate 2g, KH ₂ PO ₄ 1.333g, 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 O 0.1g, 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 6g, Tween-80 1ml, vitamin B ₁ 10μg, vitamin B ₂ 5μg, vitamin B ₆ 5μg, add water to 1L, at 8 lbs Autoclave for 30 minutes.

Pycnoporus sanguineus (L: Fr) Murr. (MK528)) was inoculated onto solid seed media. Culture at 30°C for 6-8 days. Get the bacterium block (diameter 1cm, 10 blocks) on the edge of the colony, inoculate into a Erlenmeyer flask filled with 100ml of high-pressure moist heat sterilized fermentation medium, and cultivate in the dark at 30°C and 200rpm shaker. The inducer 2,5-dimethylaniline (2,5-Dimethylaniline) was added to the 4th day of culture to make the final concentration 10 μM, and cultured for another 8 days, the fermentation broth was collected, and the enzyme activity was measured to be 63U/ml.

2. Extraction of laccase

Take the fermentation broth, centrifuge at 5000rpm to remove the fermentation sediment, take the supernatant and vacuum filter, and ultrafiltrate the obtained filtrate through a PLGC membrane (10000D) to collect the ultrafiltration concentrate. Adopt two-step ammonium sulfate precipitation method precipitation concentrated solution subsequently: the first step is with the saturated ammonium sulfate of 35% (W/V) at 4 ℃ and is precipitated by stirring, and 10000rpm centrifuges 1 hour, discards precipitation, stays supernatant; Add 80% (W/V) saturated ammonium sulfate to the supernatant obtained in the first step, stir and precipitate at 4°C, let it stand at 4°C for 2 hours, then centrifuge at 10000rpm for 1 hour, discard the supernatant, and leave the precipitate. The precipitate obtained in the second step was dissolved with twice the volume of 100mM potassium phosphate buffer (pH 5.7), and dialyzed overnight in 10mM potassium phosphate buffer to obtain a crude laccase preparation. Wherein the ultrafiltration step yield rate is 81.82%, the ammonium sulfate precipitation step yield rate is 88.89%, and the potassium phosphate buffer solution dialysis yield rate is 96.46%.

Take 10ml of the obtained crude laccase preparation for purification by ion exchange column chromatography. The ion exchange medium is DEAESepharose F.F., and the equilibrium buffer is 20mM histidine buffer (pH6.0). The elution process adopts gradient elution, the gradient is 0.1-0.6M NaCl histidine eluent, and the flow rate is 1mL/min. The collected laccase part was passed through Sephadex G-100, the eluent used was water, and the flow rate was 0.5 mL/min. Then freeze-dry the collected laccase solution to obtain blue laccase.

In this example, the determination of laccase activity uses the internationally accepted 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS){2,2'-azino- bis-[3-ethylthiazoline-6-sulphonate]} method to determine.

The calculation formula of enzyme activity is U=ΔA·V/(ε·L·T)

In the above formula, ΔA represents the change in the absorbance value of the UV spectrophotometer, 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 the oxidized ABTS, L is the light path of the cuvette (unit: cm), and 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. 500 μL of 50 mM tartrate buffer (pH 4), 390 μL of water, 100 μL of 500 μM ABTS and 10 μL of fermentation broth contained in a volume of 1 mL of the reaction system, the selected wavelength is 420 nm. If the laccase activity in the fermentation broth is too high, it can be diluted appropriately.

Claims (6)

1. Pycnoporus sanguineus strain mk528, whose preservation number is CGMCC №1124 in the General Microbiology Center of China Microbiological Culture Collection Management Committee. 2. A method for producing laccase, comprising the step of fermenting and cultivating the Miscella haemopore mk528 described in claim 1 under aerobic and light-shielding conditions, and extracting the laccase from the fermentation medium; The medium includes maltose, ammonium tartrate and inorganic components, and 2,5-dimethylaniline is added to the medium after 3-5 days of fermentation; the extraction of laccase from the fermentation medium includes the following steps: centrifugal separation, Ultrafiltration concentration, ammonium sulfate fractional precipitation, ion exchange column separation and gel filtration column separation. 3. The method according to claim 2, characterized in that: the inorganic components in the culture medium include ferrous sulfate, magnesium sulfate, manganese sulfate, zinc sulfate, copper sulfate, calcium chloride and cobalt chloride. 4. The method according to claim 2 or 3, characterized in that: the fermentation medium is composed of the following substances, maltose: 1.0-2.0%; ammonium tartrate: 0.1-0.3%, KH ₂ PO ₄ : 0.132-0.134 %; NaH ₂ PO ₄ ·12H ₂ O: 0.038-0.040%; MgSO ₄ ·7H ₂ O: 0.04-0.06%; Sodium succinate (CH ₂ COONa) ₂ ·6H ₂ O: 0.117-0.119%; FeSO ₄ · 7H ₂ O: 0.00314-0.00316%; CaCl ₂ 2H ₂ O: 0.009-0.011%; MnSO ₄ H ₂ O: 0.0034-0.0036%; CH ₃ COONa 3H ₂ O: 0.0407-0.0409%; CoCl ₂ 6H ₂ O: 0.005-0.007%; ZnSO ₄ 7H ₂ O: 0.0027-0.0029%; CuSO ₄ 5H ₂ O: 0.0167-0.0169%; Wheat Bran 5-7g; Tween-80: 0.9-1.1ml; Vitamin B _{Vitamin B 1} : 10 μg; Vitamin B ₂ : 5 μg; Vitamin B ₆ : 5 μg, add water to 1 liter, wherein the percentage is the percentage of the weight of solids based on water to the volume of water. 5. The method according to claim 2 or 3, characterized in that the amount of 2,5-dimethylaniline added is 10 μM. 6. The method according to claim 2 or 3, characterized in that: the ultrafiltration concentration is ultrafiltration with an ultrafiltration column with a molecular weight cut-off of 10,000 Daltons; the ammonium sulfate fractional precipitation adopts two-step ammonium sulfate precipitation Ammonium sulfate saturation is 30% to 40% and 70% to 90%, respectively.