CN102080046A - High-yield laccase strain and method for producing laccase through fermentation - Google Patents

Abstract

The invention provides a pycnoporus strain (Pycnoporus sp.SYBC-L3) of high-yield laccase, which was collected in the China Center for Type Culture Collection on September 15, 2010, with a collection number of CCTCC NO:M 2010235. Simultaneously, a method for producing the laccase through fermentation is invented, wherein the pycnoporus strain is used as a starting strain, seed culture is performed, soybean meal (or sodium nitrate) is used as a nitrogen source and malt sugar (or glucose, fructose and cellobiose) and the like are used as a carbon source; and the highest enzyme activity of the fermentation broth can be over 60,000 U/L. The produced laccase can be widely applied in the fields of food, agriculture, industrial wastewater treatment and the like, and belongs to the field of biotechnology.

Description

A high-yield laccase strain and method for fermenting and producing laccase

technical field

The present invention utilizes a bacterial strain (Pycnoporus sp.SYBC-L3) with high yield of laccase and utilizes the bacterial strain through seed cultivation and uses soybean meal (or sodium nitrate) as a nitrogen source, maltose (or glucose, fructose, cellobiose) etc. The invention relates to a method for fermenting and producing laccase from a carbon source, and the prepared enzyme can be applied to food, agriculture, animal husbandry, industry and environmental waste water treatment, etc., and belongs to the field of biotechnology. the

Background technique

Laccase (Laccase, EC 1.10.3.2) is a copper-containing polyphenol oxidase with a wide range of substrates. It can not only catalyze the oxidation of a variety of aromatic compounds, but also degrade lignin and remove many toxic phenolic substances such as The toxicity of phenoxy herbicides can also decolorize various dyes and remove the toxicity of petroleum industry wastewater. Therefore, it has great research in the fields of papermaking, environmental protection, food, medicine and hygiene, biological detection, and improvement of lawn soil structure. and application value. the

Laccases are widely distributed in fungi, plants, animals, bacteria and insects. However, white-rot fungi of the Basidiomycotina subphylum are mainly used for the production of laccase. the

Existing studies have shown that laccase has great potential for applications in pulping, fabric decolorization, biosensors, food industry, and environmental protection. the

Contents of the invention

The present invention screens and provides a high-yield laccase wild bacterial strain (Pycnoporus sp.SYBC-L3) and utilizes the bacterial strain through seed cultivation and using soybean meal (or sodium nitrate) as a nitrogen source and maltose (or glucose, fructose, fiber disaccharide) etc. are carbon source fermentation methods for producing laccase, and the prepared laccase can be widely used in food, agriculture, animal husbandry, industry and environmental wastewater treatment, etc., to replace expensive imported laccase. the

Technical solution of the present invention: a high-yielding laccase-producing Mycoporus strain named Pycnoporus sp.SYBC-L3, which has been preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M2010235, and a preservation date of 2010 September 15. the

Using this bacterium as the starting strain, the laccase is produced by seed cultivation and fermentation with soybean meal (or sodium nitrate) as the nitrogen source and maltose (or glucose, fructose, cellobiose) as the carbon source; the process is as follows:

(1) Seed cultivation

Seed culture medium: in terms of mass volume concentration: 5% glucose, 1% yeast juice, 0.5% peptone. Inoculation process: Take a ring of slant strains and spot mycelia on a PDA plate, culture at 30°C for 7 days, take a plate and wash it with 10ml sterile water, and break it into a uniform spore suspension. Take 1ml of the spore suspension to inoculate Put it into a 250ml Erlenmeyer flask, and the filling volume is 50ml. the

Cultivation conditions: rotation speed 200rpm, temperature 30 degrees, constant temperature cultivation for 2 days, that is, the seeds fermented to produce enzymes in the fermenter. the

(2) Enzyme production culture

Enzyme production medium: medium (1L): soybean meal (or sodium nitrate) 6g, maltose (or glucose, fructose, cellobiose) 12g, copper sulfate pentahydrate 0.4g, potassium dihydrogen phosphate 1g, disodium hydrogen phosphate 0.2 g, magnesium sulfate heptahydrate 0.5g, manganese sulfate 0.034g. Initial pH: natural. the

Fermentation tank: The specification is a capacity of 5L. The actual liquid volume of the fermented enzyme production medium is 3L

Fermentation parameters: speed 200r/min, temperature 30 degrees, ventilation 1.4L/min, 120h. the

According to the above conditions, the fermentation is carried out in a 5L fermenter, and the laccase activity of the fermentation broth is up to 60000U/L or more. the

The present invention screens out a high-yielding laccase-producing wild Mycoporus strain from dead wood collected in Guangdong Province, and names it as Pycnoporus sp. SYBC-L3. the

1. Screening of this strain:

1.1 flat plate primary screening

Sample treatment method: Soak the material in a small amount of sterile water at 30°C for 24h, then dilute it to 10 ^-8 by gradient dilution method, take 1mL and evenly coat it with guaiacol (α-naphthol) sterilized at 121°C for 20min ) 0.04% PDA (20% potato, 2% glucose, 2% agar) plate culture medium at a constant temperature of 30°C for 3 days, and pick the colonies with red color-changing circles around them and separate them by streaking. Re-inoculate the isolated strains capable of producing chromatic circles on a new plate, and record the diameter of the chromatic circles, screen out strains with higher laccase-producing activity and store them for later use.

1.2 shake flask re-screening

Rinse the plate covered with hyphae with 10 mL of sterile water, pipette 1 mL into the seed medium (5% glucose, 1% yeast extract, 0.5% peptone, natural pH, sterilized at 121°C for 20 minutes), and fill the seed medium with liquid The amount is 50mL culture medium in 250mL Erlenmeyer flask, culture temperature 30 ℃, rotating speed 200r/min shaker constant temperature culture 48 hours, take bacterial suspension (according to inoculum size 10%) insert liquid state fermentation basal medium (glucose 1%, Ammonium tartrate 0.4%, KH ₂ PO ₄ 0.1%, Na ₂ HPO ₄ 0.02%, MgSO ₄ 7H ₂ O 0.05%, CuSO ₄ 0.0007%, MnSO ₄ 0.0034%, sterilized at 121°C for 20min), liquid fermentation basal medium The filling volume is 50mL medium in a 250mL Erlenmeyer flask, the culture temperature is 30°C, the rotation speed is 200r/min, and the shaker is kept at constant temperature for 8 days.

1.3 After screening by the above two methods, a strain with relatively high laccase activity was finally screened, plated colonies (Figure 1), electron microscopy (Figure 2.), 16S rDNA sequence identification (see sequence.) and identified as dense pores The genus is named Pycnoporus SP.SYBC-L3. the

2. Determination method of enzyme activity

The 3mL reaction system includes 0.5ml10mmol/LDMP (2,6-dimethoxyphenol), 2.4ml0.1mol/L disodium hydrogen phosphate-citrin buffer solution (pH3.5), 0.1ml crude enzyme solution (fermentation broth Centrifuge at 12,000g in a refrigerated high-speed centrifuge at 4°C for 10 minutes to obtain the supernatant as the crude enzyme solution (diluted depending on the activity), replace the crude enzyme solution with deionized water as a blank control, and measure the absorbance at 469nm every 10s Read the absorbance value once, and generally stop the measurement after 1 min, and calculate the enzyme activity based on the linear range of the reaction. Definition of enzyme activity: the amount of enzyme required to catalyze one micromole of substrate in one minute, and the enzyme activity is expressed in U·L ^-1 .

Beneficial effects of the present invention:

Firstly, a laccase-producing bacterial strain and a method for preparing laccase by liquid fermentation are provided by using the bacterium as a starting strain. Compared with other bacterial strains and the method for preparing laccase, the bacterial classification and fermentation enzyme production method of the present invention have the following characteristics:

1. The fermentation performance is stable, and the bacteria are easy to cultivate. 

2. The enzyme production cycle is short, and the fermentation can be stopped on the 5th day. 

3. High enzyme activity, up to 60000U/L or more. 

4. The laccase content in the crude enzyme solution accounts for more than 80% of the total protein content, and it becomes the dominant product outside the cell, which is convenient for separation and purification. 

5. The crude enzyme is stable in nature. When DMP is used as a substrate, it can exert its maximum catalytic effect in an acidic environment. The optimum temperature is 55 degrees, which is relatively heat-resistant. the

6. It has a wide range of substrates and has broad application prospects. 

Therefore the bacterial strain of the present invention and the laccase produced by the method for producing laccase by fermentation can be widely used in aspects such as food, agriculture, animal husbandry, industry and environmental waste water treatment, and can replace expensive imported laccase, so the present invention The strain and fermentative enzyme production method have extensive industrial use value and remarkable economic benefit prospect. the

Description of attached chart

Figure 1 and Figure 2 are the plate colony and scanning electron microscope morphology of Pycnoporus sp.SYBC-L3 strain, respectively. the

Biological Material Sample Preservation

Pycnoporus sp.SYBC-L3 has been preserved in the China Center for Type Culture Collection, the preservation number is: CCTCC NO: M 2010235, and the preservation date is September 15, 2010.

Detailed ways

Example 1

Take a ring of slant strains and spot mycelium on a PDA plate, culture at 30°C for 7 days, take a plate and wash it with 10ml sterile water, and break it into a uniform spore suspension. Add 50ml seeds per 1ml spore suspension Medium (250ml Erlenmeyer flask), prepare several copies. Cultivation conditions: rotation speed 200rpm, temperature 30 degrees, constant temperature cultivation for 2 days, that is, the seeds that ferment and produce enzymes. the

Put 3L of enzyme-producing medium in a 5L fermenter, add the above-mentioned fermented enzyme-producing seeds according to 10% inoculum, the culture conditions are: rotation speed 200r/min, temperature 30°C, ventilation rate 1.4L/hour, culture time 120h, The enzyme activity of the fermentation broth reaches 60200U/L. the

Enzyme production medium is soybean meal 18g, maltose 36g, copper sulfate pentahydrate 1.2g, potassium dihydrogen phosphate 3g, disodium hydrogen phosphate 0.6g, magnesium sulfate heptahydrate 1.5g, manganese sulfate 0.102g. Initial pH: natural. the

Example 2

The same example 1 was used for the cultivation of fermented enzyme-producing seeds. the

Put 3L of enzyme-producing medium in a 5L fermenter, add the above-mentioned fermented enzyme-producing seeds according to 10% inoculum, the culture conditions are: rotation speed 200r/min, temperature 30°C, ventilation rate 1.4L/hour, culture time 120h, The enzyme activity of the fermentation broth reached 48200U/L. the

Enzyme production medium is soybean meal 18g, glucose 36g, copper sulfate pentahydrate 1.2g, potassium dihydrogen phosphate 3g, disodium hydrogen phosphate 0.6g, magnesium sulfate heptahydrate 1.5g, manganese sulfate 0.102g. Initial pH: natural. the

Example 3

The same example 1 was used for the cultivation of fermented enzyme-producing seeds. the

Put 3L of enzyme-producing medium in a 5L fermenter, add the above-mentioned fermented enzyme-producing seeds according to 10% inoculum, the culture conditions are: rotation speed 200r/min, temperature 30°C, ventilation rate 1.4L/hour, culture time 120h, The enzyme activity of the fermentation broth reaches 53000U/L. the

Enzyme production medium is 18g of soybean meal, 36g of fructose, 1.2g of copper sulfate pentahydrate, 3g of potassium dihydrogen phosphate, 0.6g of disodium hydrogen phosphate, 1.5g of magnesium sulfate heptahydrate, and 0.102g of manganese sulfate. Initial pH: natural. the

Example 4

The same example 1 was used for the cultivation of fermented enzyme-producing seeds. the

Put 3L of enzyme-producing medium in a 5L fermenter, add the above-mentioned fermented enzyme-producing seeds according to 10% inoculum, the culture conditions are: rotation speed 200r/min, temperature 30°C, ventilation rate 1.4L/hour, culture time 120h, The enzyme activity of the fermentation broth reached 51800U/L. the

Enzyme production medium is 18g of sodium nitrate, 36g of maltose, 1.2g of copper sulfate pentahydrate, 3g of potassium dihydrogen phosphate, 0.6g of disodium hydrogen phosphate, 1.5g of magnesium sulfate heptahydrate, and 0.102g of manganese sulfate. Initial pH: natural. the

sequence listing

Claims (2)

1. the samguineus of plant height product laccase belongs to bacterial strain, and called after Pycnoporus sp.SYBC-L3 has been stored in Chinese typical culture collection center, and deposit number is CCTCC NO:M 2010235, preservation date on 09 15th, 2010.

2. the method for using the described strain fermentation of claim 1 to produce laccase, it is characterized in that being the bacterial classification that sets out with this bacterium, through seed culture with dregs of beans (or SODIUMNITRATE) is nitrogenous source, is that carbon source through fermentation is produced laccase with maltose (or glucose, fructose, cellobiose) etc., and technology is:

(1) seed culture

Seed culture medium: in the quality volumetric concentration: 5% glucose, 1% yeast water, 0.5% peptone.

Inoculating process: get slant strains one ring point on the PDA flat board and connect mycelia, cultivated 7 days for 30 ℃, get a flat board and use the 10ml aseptic water washing, and be broken into uniform spore suspension. get the triangular flask that the 1ml spore suspension inserts the 250ml specification, liquid amount is 50ml.

Culture condition: rotating speed 200rpm, 30 ℃ of temperature, constant temperature culture 2 days is the seed of enzymatic production.

(2) producing enzyme cultivates

Produce the enzyme substratum: the 1L substratum calculates: dregs of beans (or SODIUMNITRATE) 6g, maltose (or glucose, fructose, cellobiose) 12g, cupric sulfate pentahydrate 0.4g, potassium primary phosphate 1g, Sodium phosphate dibasic 0.2g, magnesium sulfate heptahydrate 0.5g, the initial pH of manganous sulfate 0.034g.: nature.

Fermentor tank: specification is the 5L capacity. it is 3L that real attenuation produces enzyme substratum liquid amount.

Fermentation parameter: inoculum size 10%, rotating speed 200r/min. temperature 30 degree. air flow 1.4L/min. incubation time 5-8 days.

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