CN105505901A - Composition for inducing fungi to realize high-yield production of cellulase and application method thereof - Google Patents

Abstract

The invention relates to the field of cellulase fermentation, in particular to a composition for inducing high-yield cellulase in fungi and an application method thereof. The invention discloses a composition for inducing high-production cellulase of fungi, which comprises the following components in parts by weight: 0.5-1.5 parts of lignocellulose and 6-9.5 parts of microcrystalline cellulose. The method of using the composition for inducing high-yield cellulase in fungi is as follows: adding the composition to fungal culture medium, and the added amount of the composition is 30-35 g/L. The composition for inducing fungi to produce cellulase is simple in proportion, easy to obtain raw materials, and low in cost, and is a novel and cheap inducer for cellulase synthesis; the use method of the composition has the advantages of simple process, mild conditions and The advantages of environmental friendliness provide a new way for the high-efficiency and low-cost production of cellulase.

Description

A composition for inducing high-yield cellulase in fungi and its application method

technical field

The invention relates to the field of cellulase fermentation, in particular to a composition for inducing high-yield cellulase in fungi and an application method thereof.

Background technique

Carbon source is the primary factor affecting cellulase gene expression, and fungal cellulase needs to be induced in the presence of cellulose substrates. Natural cellulose raw materials are the best substrates for fungal cellulase, which can effectively promote the production of cellulase. The natural cellulose raw material itself as an insoluble carbon source does not directly trigger the induction of cellulase, but releases soluble oligosaccharides through the hydrolysis of cellulose by the cellulase expressed at the basal level, and the oligosaccharides enter the cell to induce cellulose Enzymes are abundantly expressed. At present, many oligosaccharides and their derivatives have been studied, and it is found that cellobiose, sophorose, gentiobiose, lactose, etc. have an inducing effect on the production of cellulase. However, adding this oligosaccharide directly into the culture medium to induce fungi to produce cellulase is too costly, and disaccharides have a feedback inhibitory effect on cellulase, affecting the degradation of cellulase. Therefore, it is inappropriate to use disaccharide substances as inducers of cellulase production in the medium. At present, it is urgent to find new and cheap inducers of cellulase synthesis, so as to provide a new way for efficient and low-cost production of cellulase.

Contents of the invention

One of the objectives of the present invention is to provide a composition for inducing high-yield cellulase in fungi, which is used to solve the problems of high cost of cellulase induction and general effects of inducers. The second object of the present invention is to provide a method for using the composition for inducing high-yielding fungal cellulase, which is used to provide a better method for using the composition for inducing high-yielding fungal cellulase according to the present invention, and to play a role in inducing high-yielding fungal cellulose. Enzyme composition for best results.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A composition for inducing high-production cellulase in fungi is characterized in that it comprises the following components in parts by weight: 0.5-1.5 parts of lignocellulose and 6-9.5 parts of microcrystalline cellulose.

Preferably, the following components are included in parts by weight: 1-1.5 lignocellulose and 7-8 microcrystalline cellulose.

Preferably, the following components are included in parts by weight: lignocellulose 1, and microcrystalline cellulose 7.5.

Preferably, the lignocellulose is any one of laver, rice or wheat.

Preferably, the lignocellulose is laver.

A method for using a composition for inducing high production of cellulase in fungi, characterized in that the composition is added to a fungal culture medium, and the amount of the composition added is 30-35 g/L.

Preferably, the added amount of the composition is 33g/L.

Preferably, the fungal culture medium is a liquid culture medium, and the liquid culture medium also includes nitrogen sources and inorganic salt ions required for fungal growth: a mass fraction of 1.7% corn steep liquor dry powder, a mass fraction of 0.5% ( NH ₄ ) ₂ SO ₄ , MgSO ₄ with a mass fraction of 0.1%, glycerin with a mass fraction of 0.25%, and CaCO ₃ with a mass fraction of 0.25%, and adjust the pH to 4.5-5.5.

Preferably, the following steps are also included:

The spore suspension of the fungus is inserted into the liquid medium, and cultured for 96-120 hours on a shaker at a speed of 170-200 rpm under the condition of 28-30°C.

Preferably, the fungus is one of Penicillium juniperus or Trichoderma reesei.

The beneficial effects of the present invention are:

The method of the invention adopts the ratio matching of lignocellulose and microcrystalline cellulose to realize full utilization of lignocellulose and save resources;

Lignocellulose is any one of laver, rice or wheat. It is a common and easy-to-obtain resource with low cost and solves the problem of lignocellulose processing in daily life;

The polysaccharides produced by the hydrolysis of lignocellulose provide raw materials for fungi to proliferate in large numbers and induce their synthesis of cellulase, replacing the practice of directly adding disaccharides to the medium as inducers for cellulase production, reducing production costs, and also Feedback inhibition of cellulase does not occur, nor does it affect cellulase degradation.

In a word, the composition for inducing fungi to produce cellulase according to the present invention is simple in proportion, easy to obtain raw materials, and low in cost, and is a novel and cheap inducer for cellulase synthesis; The advantages of mildness and environmental friendliness provide a new way for the efficient and low-cost production of cellulase.

detailed description

The present invention will be further described in detail below in conjunction with the embodiments, so that those skilled in the art can implement it with reference to the description.

Example 1

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sieved laver according to the ratio of 9.5:0.5, and add the composition of inducing fungal cellulase production into the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 30 g /L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Inoculate the spore suspension of Penicillium piceum into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermentation liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in the Penicillium juniperi enzyme solution to increase by 46.2%. The protein concentration increased by 35.7%, the exonuclease activity increased by 47.6%, and the filter paper enzyme activity increased by 14.2%.

Example 2

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sieved laver at a ratio of 9:1, and add it into the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 33 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Inoculate the spore suspension of Penicillium piceum into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermentation liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in the Penicillium juniperi enzyme solution to increase by 61.2%. Protein concentration increased by 56.7%, exonuclease activity increased by 61.4%, and filter paper enzyme activity increased by 19.5%.

Example 3

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sieved laver according to 8.5:1, and add it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 35 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerin 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Inoculate the spore suspension of Penicillium piceum into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermentation liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in Penicillium juniperi enzyme solution to increase by 67.9%. The protein concentration increased by 59.1%, the exonuclease enzyme activity increased by 100%, and the filter paper enzyme activity increased by 39.2%.

Example 4

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2, mixing the microcrystalline cellulose and the sieved laver according to 7.5:1.5, and adding it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 30 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerin 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Inoculate the spore suspension of Penicillium piceum into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermentation liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in the Penicillium juniperi enzyme solution to increase by 57.2%. The protein concentration increased by 54.7%, the exonuclease activity increased by 70.0%, and the filter paper enzyme activity increased by 19.3%.

Example 5

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sieved laver according to 6:0.5, and add it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 33g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Inoculate the spore suspension of Penicillium piceum into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermentation liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in the Penicillium juniperi enzyme solution to increase by 21.2%. The protein concentration increased by 14.2%, the exonuclease enzyme activity increased by 24.5%, and the filter paper enzyme activity increased by 11.6%.

Example 6

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sieved laver according to 9:1, and add it into the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 35g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Insert the spore suspension of Trichoderma reesei (T. reesei) into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermented liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in Trichoderma reesei enzyme solution to increase by 104%. Protein concentration increased by 24%, exonuclease activity increased by 17.2%, and xylanase activity increased by 479%.

Example 7

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2, mixing the microcrystalline cellulose and the sieved laver according to 7.5:1, and adding it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 30 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Insert the spore suspension of Trichoderma reesei (T. reesei) into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermented liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in Trichoderma reesei enzyme solution to increase by 112%. Protein concentration increased by 21.4%, exonuclease activity increased by 54.1%, and xylanase activity increased by 521%.

Example 8

Step 1, drying the laver to a constant weight, then pulverizing it with a pulverizer, and sieving the pulverized laver through a 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sieved laver according to 8.5:1.5, and add it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 33g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Insert the spore suspension of Trichoderma reesei (T. reesei) into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermented liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces the enzyme activity of beta-glucosidase in Trichoderma reesei enzyme solution to increase by 108%. The protein concentration is increased by 15.7%, the exonuclease activity is increased by 50%, and the xylanase activity is increased by 495%.

Example 9

Step 1, dry the rice stalks to a constant weight, then pulverize them with a pulverizer, and sieve the pulverized rice with an 80-mesh standard sieve for subsequent use;

Step 2, mixing the microcrystalline cellulose and the sieved rice stalk at a ratio of 9:1, and adding it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 35 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerol 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Insert the spore suspension of Trichoderma reesei (T. reesei) into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermented liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces a 59.5% increase in filter paper enzyme activity in the Trichoderma reesei enzyme solution. The enzymatic activity of endonuclease is increased by 72.5%, and that of exonuclease is increased by 103.5%.

Example 10

Step 1, dry the wheat stalks to constant weight, then pulverize them with a pulverizer, and sieve the pulverized rice through an 80-mesh standard sieve for subsequent use;

Step 2, mixing the microcrystalline cellulose and the sieved wheat straw at a ratio of 9:1, adding it to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 30 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerin 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Insert the spore suspension of Trichoderma reesei (T. reesei) into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermented liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces a 41.3% increase in filter paper enzyme activity in the Trichoderma reesei enzyme solution. The enzyme activity of endonuclease was increased by 68.7%.

Example 11

Step 1, drying Miscanthus stalks to constant weight, then pulverizing them with a pulverizer, and sieving the pulverized paddy rice with an 80-mesh standard sieve for subsequent use;

Step 2. Mix the microcrystalline cellulose and the sifted Miscanthus stalks at a ratio of 9:1, and add them to the fungal liquid culture medium as a composition for inducing fungal cellulase production, and the addition amount is 33 g/L;

Step 3, adding N sources and inorganic salt ions needed for fungal growth to the liquid medium, corn steep liquor dry powder 1.7%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ 0.1%, glycerin 0.25%, CaCO30.25%, Adjust pH4.5-5.5;

Step 4: Insert the spore suspension of Trichoderma reesei (T. reesei) into the culture medium, and cultivate for 96-120 hours at 30° C. on a shaking table with a rotation speed of 170-200 rpm.

After 120 hours of fermentation, the enzyme activity of the fermented liquid was measured by the international standard method recommended by the International Union of Theoretical and Applied Chemistry (IUPAC). The composition induces a 38.4% increase in the filter paper enzyme activity and a 72.1% increase in the exonuclease activity in the Trichoderma reesei enzyme solution.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and embodiments shown here, without departing from the general concept defined by the claims and their equivalents.

Claims (10)

1. A composition for inducing high-yield cellulase in fungi, characterized in that it comprises the following components in parts by weight: 0.5-1.5 parts of lignocellulose and 6-9.5 parts of microcrystalline cellulose. 2 . The composition for inducing high-yield cellulase in fungi according to claim 1 , characterized in that it comprises the following components in parts by weight: lignocellulose 1-1.5, and microcrystalline cellulose 7-8. 3. The composition for inducing high-yield cellulase in fungi according to claim 2, characterized in that it comprises the following components in parts by weight: lignocellulose 1, and microcrystalline cellulose 7.5. 4. The composition for inducing high-yield cellulase in fungi according to claim 3, wherein the lignocellulose is any one of laver, rice, wheat or Miscanthus. 5. The composition for inducing high-yield cellulase in fungi as claimed in claim 4, wherein the lignocellulose is laver. 6. A method for using the composition of inducing fungal high-yield cellulase as claimed in claim 1, wherein the composition is added to the fungal culture medium, and the amount of the composition added is 30-35g /L. 7. The method for using the composition for inducing high-yield cellulase in fungi according to claim 6, wherein the amount of the composition added is 33 g/L. 8. the use method of the composition of inducing fungal high-yield cellulase as claimed in claim 7, it is characterized in that, described fungal culture medium is liquid culture medium, and described liquid culture medium also comprises the nitrogen source needed for fungal growth and inorganic The salt ions are: corn steep liquor dry powder with a mass fraction of 1.7%, (NH ₄ ) ₂ SO ₄ with a mass fraction of 0.5%, MgSO ₄ with a mass fraction of 0.1%, glycerol with a mass fraction of 0.25%, and a mass fraction of 0.25% CaCO ₃ , and adjust the pH to 4.5-5.5. 9. the use method of the composition of inducing fungal high-yield cellulase as claimed in claim 8, is characterized in that, also comprises the following steps: The spore suspension of the fungus is inserted into the liquid medium, and cultured for 96-120 hours on a shaker at a speed of 170-200 rpm under the condition of 28-30°C. 10. The method for using the composition for inducing high-yield cellulase in fungi according to claim 9, wherein the fungus is one of Penicillium juniperus or Trichoderma reesei.