CN111500559A - A kind of cellulase fermentation regulator and application - Google Patents

Abstract

The invention discloses a cellulase fermentation regulator and application. The cellulase fermentation regulator is an inorganic ammonium salt. The present invention provides a new use of ammonium salt in cellulase fermentation, and is an environment-friendly technology for recycling and harmless utilization of straw.

Description

A kind of cellulase fermentation regulator and application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a cellulase fermentation regulator and application.

Background technique

Cellulose is a glucan formed by connecting glucose molecules through β-1,4-glycosidic bonds, usually containing thousands of glucose units. It is the most abundant organic substance on earth and the main component of plant cell walls; fiber Cellulose is the most widely distributed and most abundant polysaccharide in nature. More than half of the carbon in the plant kingdom exists in cellulose; plants produce the most abundant and cheapest cellulose resources on earth through photosynthesis. Plants produce up to 150-200 billion tons of dry matter, of which the total amount of cellulose and hemicellulose is 85 billion tons, accounting for more than 50% of the carbon content of the plant kingdom.

Cellulase is a general term for a group of enzymes that degrade cellulose to generate glucose. It is not a single enzyme, but a multi-component enzyme system that acts synergistically; including: endoglucanase (1,4-β- D-glucan glucanohydrolase), this type of enzyme generally acts on the non-crystalline region inside cellulose, randomly hydrolyzes β-1,4-glycosidic bonds, truncates long-chain cellulose molecules, and produces a large number of small molecules with non-reducing ends Cellulose; exo-glucanase (1,4-β-D-glucancellobiohydrolase), which acts on the end of cellulose linear molecules, hydrolyzes the β-1,4-glycosidic bond, and cleaves one fiber at a time Sugar molecules, it is also called cellobiohydrolase (cellobiohydrolase); β-glucosidase (β-glucosidase), these enzymes generally hydrolyze cellobiose into glucose molecules.

The sources of cellulase are very wide, and insects, mollusks, protozoa, bacteria, actinomycetes and fungi can all produce cellulase; but the strains with strong effect on cellulose are mostly Trichoderma, Aspergillus Strains of the genera Aspergillus, Penicillium, Pellienlalia and Acremonium, especially T. virde and its relatives.

Cellulase is a group of inducible enzymes, which are regulated by multiple factors including inducers in the environment, genes, promoters and repressors of the bacteria itself.

Carbon signal studies have shown that lactose, cellobiose, etc. induce Trichoderma reesei to express cellulase, which can positively regulate cellulase gene expression; using cellulose as a carbon source can only enhance the cellulase expression of Trichoderma reesei, but with cellulose as a carbon source. When lactose was the carbon source, light could inhibit cellulase expression; BLR1 and BLR2 were blue light receptor proteins in Trichoderma reesei; BLR1 and BLR2 positively regulated cellulase gene transcription under light conditions.

Many proteins can bind to the promoter regions of cellulase and hemicellulase genes to activate or inhibit the expression of related genes; Xyrl, Ace2, Hap2/3/5, Acel and Crel were discovered earlier to regulate cellulase expression. transcription factor. Ace2 plays a role in the regulation of xyn2 gene: inhibits early induction of xyn2; enhances persistent expression of xyn2 in late stage.

The Gα part of the eukaryotic G protein component is involved in the expression of cellulase genes, and the transcription of cellulase in the double deletion strains of gna1 and gna3 is inhibited under certain conditions; The secretion of endoglycanase nearly doubled, and cAMP regulates the expression of Trichoderma reesei cellulase gene in a carbon source-dependent manner.

SUMMARY OF THE INVENTION

The invention aims to overcome the deficiencies of the prior art, and provides a cellulase fermentation regulator and application.

In order to achieve the above object, the technical scheme provided by the invention is:

The cellulase fermentation regulator is an inorganic ammonium salt.

Preferably, the inorganic ammonium salt includes at least one of ammonium sulfate, ammonium hydrogen sulfate, ammonium chloride, ammonium nitrate, ammonium carbonate, and ammonium hydrogen carbonate.

The ammonium salt is used as a cellulase fermentation regulator, or used to prepare a cellulase fermentation regulator. When the ammonium salt is used as a cellulase fermentation regulator, when the fermentation medium is a solid medium, the addition amount of the ammonium salt is 2.5%-3.5% of the weight of the solid medium; when the fermentation medium is a liquid medium, the ammonium salt is added. The added amount is 1%-3% of the liquid medium weight.

The process of using ammonium salt for cellulase fermentation regulation and production is as follows: using fungal cellulase-producing bacteria (which can be Trichoderma kronesii, Trichoderma viride, Penicillium, Aspergillus aculeatus, etc.) as strains, and prepared by strains, Solid or liquid fermentation, combined with appropriate concentration of inorganic ammonium salt regulation, the experimental results show that the level of cellulase fermentation increased by more than 200%.

The present invention is further described below in conjunction with principle:

The principle that described ammonium salt regulates cellulase fermentation is summarized as follows:

(1) The functional group that plays a regulatory role is ammonium ion (cation), not anion (sulfate, carbonate, nitrate or chloride ion, etc.);

(2) Ammonium ions do not play a role in the nutrition of nitrogen sources during fermentation regulation, and cannot promote the increase in biomass of cellulase-producing bacteria. On the contrary, when ammonium ions play a regulating role, the biomass of bacteria decreases, but The total amount of cellulase protein is significantly increased, and the cellulase activity is significantly improved; compared with the process without ammonium salt regulation, the cellulase activity is significantly different, and is increased by 2-6 times;

(3) The regulation of ammonium ions is the regulation at the gene level. By significantly increasing the activity of cellulase transcription factors and promoters, the transcription level of cellulase genes is improved, thereby improving the translation level of cellulase and realizing the total cellulase level. increase in volume;

(4) The regulating effect of ammonium ions depends on the existence of carbon sources (cellulose powder, etc.), and a certain carbon source is required in the medium; it has the characteristics similar to cellulose for cellulase induction, but ammonium ions have the same effect on fiber. The "induction" effect of nephelase is much higher than that of cellulose itself.

The present invention can achieve a significant positive regulation effect when using wild strains to ferment cellulase, and has significant advantages and technical progress compared with the existing genetic engineering strains with harsh regulation, complex culture medium and high cost:

(1) The medium is simple and the cost is low. The basic fermentation medium is mainly made of agricultural by-products or wastes such as bran and straw powder, which can effectively reduce the pressure of environmental pollution; the regulator is an inorganic ammonium salt, with small molecular weight, clear structure, and no Harmful, easy to obtain, low grade requirements, can be fertilizer level or industrial grade ammonium salt, compared with lactose and cellobiose, the price is extremely cheap;

(2) Ammonium salts can regulate the production of cellulase by filamentous fungi such as Trichoderma viride, Trichoderma viride, Aspergillus aculeatus, Aspergillus niger, and Penicillium. A wide range of strains;

(3) Ammonium salts are small molecules with a clear structure, which are easy to track in vivo and can be used for theoretical research on molecular mechanisms of cellulase gene expression, transcription, translation, etc. cellulase;

In a word, the present invention provides a new use of ammonium salt in cellulase fermentation, and is an environment-friendly technology for recycling and harmless utilization of straw.

Detailed ways

Example 1

Ammonium salt regulates the effect of Trichoderma kangseri AS3.2774 solid fermentation cellulase by the following steps:

Preparation of solid fermentation basic medium (control): mix 8g of 20-mesh rice straw powder, 4g of bran, and 23mL of water into a 250mL conical flask, sterilize at 121°C for 30min, and inoculate the slant of Trichoderma consei AS3.2774 after cooling The strain, the fermented rice bran koji, was cultured at 28°C for 4.5 d, during which the bran koji was shaken and ventilated once. The fermentation level of the cellulase activity of the filter paper was 0.2 IU/(g dry koji).

Preparation of solid fermentation control medium (treatment): 8.0 g of 20-mesh rice straw powder, 4.0 g of bran, 1.0 g of ammonium sulfate, 23 mL of tap water, stirred and mixed in a 250 mL conical flask, sterilized at 121 °C for 30 min, and inoculated after cooling Trichoderma kangseri AS3.2774 slanted strain, fermented rice bran koji, was cultured at 28°C for 4.5 days, during which time the bran koji was shaken and ventilated once, the fermentation level of cellulase activity of filter paper was 1.11IU/( g dried koji); the promoting effect of ammonium salt on the solid fermentation cellulase of Trichoderma kangseri AS3.2774 was increased by 5.55 times compared with the control.

Example 2

The effect of ammonium salt regulating Trichoderma viride AS3.5455 liquid fermentation cellulase is realized by the following steps:

Preparation of liquid fermentation basic medium (control): put 1.0 g of 40-mesh rice straw powder, 4.0 g of bran, and 100 mL of water in a 250-mL conical flask, sterilize at 121 °C for 25 min, and inoculate Trichoderma viride AS3.5455 slanted strain after cooling , cultured for 4 d at 28° C. under the condition of shaking table rotation speed of 130 r/min. After testing, the fermentation level of endocellulase activity was 2.3 IU/mL.

Preparation of liquid fermentation control medium (treatment): put 1.0g of 40-mesh rice straw powder, 4.0g of bran, 1.5g of ammonium chloride and 100mL of water in a 250mL conical flask, sterilize at 121°C for 25min, and inoculate Trichoderma viride AS3 after cooling The .5455 slanted strain was cultured for 4 days at 28°C and the shaker speed was 130 rpm/min. After testing, the fermentation level of endocellulase activity was 8.1IU/mL; The promoting effect of fermented cellulase was increased by 3.52 times compared with the control.

Example 3

The effect of ammonium salt regulating the liquid fermentation cellulase of Penicillium chrysogenum AS3.3871 is realized by the following steps:

Boil 8g of 20-mesh rice straw powder, 4g of bran, and 150mL of water for 20min, filter the filtrate, dilute the filtrate to 100mL, sterilize at 121°C for 30min, and inoculate Penicillium chrysogenum AS3.3871 slant strain at 28°C after cooling. After culturing for 4 days under the condition of bed speed of 150 r/min, the biomass of Penicillium chrysogenum AS3.3871 was 0.72g/L (dry weight), and the fermentation level of cellulase activity of filter paper was 0.12IU/mL.

In the same way, 8g of 20-mesh rice straw powder, 4g of bran, and 150mL of water were boiled for 20min and filtered. The filtrate was adjusted to 100mL, then 2g of ammonium nitrate was added, and sterilized at 121°C for 30min. After cooling, it was inoculated with Penicillium chrysogenum AS3.3871 slant. The strain was cultured for 4 days at 28°C and the shaker speed was 150 rpm/min. After testing, the biomass of Penicillium chrysogenum AS3.3871 was 0.67g/L, and the fermentation level of cellulase activity on the filter paper was 0.29IU/ mL; it can be seen that ammonium salt not only can not effectively promote the increase of Penicillium cell weight, but has a certain inhibitory effect; but ammonium salt significantly improves the fermentation level of Penicillium chrysogenum AS3.3871 cellulase, which is 2.16% higher than that of the control. times.

Example 4

Ammonium salt regulates the effect of Aspergillus aculeatus CGMCC No.3876 liquid fermentation cellulase by the following steps:

Preparation of liquid fermentation basic medium (control): put 1.0 g of sodium carboxymethyl cellulose, 6.0 g of bran, and 100 mL of tap water in a 250-mL conical flask, sterilize at 121 °C for 25 min, and inoculate the slant of Aspergillus aculeatus CGMCC No.3876 after cooling The strain was cultured for 4 days at 28°C and the shaking speed was 150 rpm/min. After testing, the fermentation level of endocellulase activity was 6.5IU/mL.

Preparation of liquid fermentation control medium (treatment): put 1.0 g of sodium carboxymethyl cellulose, 6.0 g of bran, 2.0 g of ammonium chloride, and 100 mL of tap water in a 250-mL conical flask, sterilize at 121 °C for 25 minutes, and inoculate Acanthopanax after cooling Aspergillus CGMCCNo.3876 slanted strain was cultured for 4 days at 28°C and the shaker speed was 150 rpm/min. After testing, the fermentation level of endocellulase activity was 21.4IU/mL; .3876 liquid fermentation cellulase has a promoting effect, which is 3.29 times higher than that of the control.

Example 5

Ammonium salt regulates the production process of Trichoderma kangseri AS3.2774 solid fermentation cellulase, and realizes through the following steps:

(1) Preparation of solid seeds: stir the bran and water in a mass ratio of 55%: 55%, and sterilize at 121-124°C for 30-40min to obtain a solid fermented bran seed medium, which is cooled in the solid fermented bran The seed culture medium was inoculated with Trichoderma kangseri AS3.2774 slanted strains, and the bran koji was incubated at 28°C for 3 days. When the white mycelium covered the surface of the bran koji, it could be used as a solid fermented seed;

(2) Preparation of solid fermentation medium: prepare fiber straw (pulverized to 10 mesh), bran, water and ammonium salt in a mass ratio of 25%:10%:61.5%:3.5%, sterilize at 121-124°C for 30 minutes -40min, standby;

(3) the fermentation production of cellulase: the solid fermentation seed is inoculated into the solid fermentation medium, the described mass ratio of the solid fermentation seed and the solid fermentation basic regulation medium is 1:50, and open fermentation is carried out, and the fermentation temperature is 22-32°C, fermentation time 5d, turn the koji once during the period, after the fermentation medium bran koji grows white mycelium, when the bran koji begins to change color and initially form spores, terminate the fermentation, extract with water, press and filter, The crude enzyme solution of cellulase was obtained, and the cellulase activity of the crude enzyme solution was measured and compared, and the endo-cellulase activity was 45.23 IU/mL;

(4) Purification and concentration of cellulase: the crude cellulase solution obtained in step (3) was pre-coated with diatomaceous earth, filtered with a second-stage plate and frame, and then concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da to prepare into a cellulase concentrate with an endo-cellulase activity of 1038IU/mL;

(5) Fine filtration, preservation and packaging of cellulase: the concentrated solution of cellulase obtained in step (4) is pre-coated with bentonite, sodium benzoate, potassium sorbate and stabilizer are added, and then the concentrated solution of cellulase prepared in step (4) is refined by a 2-stage plate and frame. Filtration to prepare an industrial-grade cellulase with an endo-cellulase activity of 1031 IU/mL, which is packaged as a finished product; the mass percentage of the sodium benzoate and cellulase concentrate is 3%, and the potassium sorbate and cellulose The mass percentage of the enzyme concentrate is 2%, and the mass percentage of the stabilizer and the cellulase concentrate is 1%.

Example 6

The production process of ammonium salt regulating Aspergillus aculeatus CGMCC No.3876 liquid fermentation cellulase is realized through the following steps:

(1) Preparation of liquid seeds in shake flasks: the bran, starch, and water are sterilized at 121° C. for 20 minutes in a mass ratio of 4%: 1.5%: 94.5% to obtain a liquid fermented bran seed medium. The bark seed medium was inoculated with Aspergillus aculeatus CGMCC No.3876 slant strain, and shaken and cultured for 2.5 days in a shaker at 32°C and a rotating speed of 180 rpm for 2.5 days, which could be used as liquid fermentation shaker seeds;

(2) Preparation of first-class seeds in liquid fermenter: prepare bran, starch, and water in a mass ratio of 4%: 1.5%: 94.5%, sterilize at 121° C. for 25 min, to obtain a first-class fermenter seed medium, which is cooled in The first-grade fermenter seed medium was inoculated with liquid fermentation shaker seeds, and stirred for 2.5 days in a fermenter with a rotation speed of 130 r/min at 32°C, which could be used as the first-grade seeds of the liquid fermenter;

(3) Preparation of secondary seeds in liquid fermenter: prepare bran, starch, and water in a mass ratio of 4%: 1.5%: 94.5%, sterilize at 121°C for 30 minutes, to obtain a secondary fermenter seed medium, which is cooled in The seed medium of the fermenter is inoculated with the first-class seeds of the liquid fermenter, and stirred and cultured for 2.5 in the fermenter at 32 ° C and the rotation speed of 150 r/min, which can be used as the second-class seeds of the liquid fermenter;

(4) Liquid cellulase-regulated fermentation production: sterilize bran, straw powder, water, and ammonium sulfate in a mass ratio of 4%: 1%: 94.5%: 2% at 121 °C for 30 minutes to obtain cellulase-regulated fermentation The culture medium was cooled in a cellulase-regulated fermentation medium, inoculated with secondary seeds in a liquid fermenter, and stirred for 4.5 days in a fermenter at 32°C, with an aeration rate of 0.5 m ³ /min and a rotational speed of 100 rpm for 4.5 days. The cellulase activity was cut to 32.44IU/mL to obtain crude cellulase enzyme solution;

(5) Purification and concentration of cellulase: the crude cellulase solution obtained in step (4) was pre-coated with diatomaceous earth, filtered with a 2-stage plate and frame, and then concentrated by an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da to prepare into a cellulase concentrate with an endo-cellulase activity of 1182IU/mL;

(6) Fine filtration, preservation and packaging of cellulase: the concentrated solution of cellulase obtained in step (5) is pre-coated with bentonite, sodium benzoate, potassium sorbate and stabilizer are added, and then the concentrated solution of cellulase obtained in step (5) is refined by a 2-stage plate and frame. Filtration to prepare an industrial-grade cellulase with an endo-cellulase activity of 1123IU/mL, which is packaged as a finished product; the mass percentage of the sodium benzoate and cellulase concentrate is 0.3%, the potassium sorbate and cellulose The mass percentage of the enzyme concentrate is 0.2%, and the mass percentage of the stabilizer and the cellulase concentrate is 0.1%.

Claims (6)

1. a cellulase fermentation regulator, is characterized in that, described cellulase fermentation regulator is inorganic ammonium salt. 2. The cellulase fermentation regulator according to claim 1, wherein the inorganic ammonium salt comprises at least one of ammonium sulfate, ammonium hydrogen sulfate, ammonium chloride, ammonium nitrate, ammonium carbonate, and ammonium hydrogen carbonate kind. 3. Use of an ammonium salt as a cellulase fermentation regulator. 4. The use according to claim 3, wherein the ammonium salt is an inorganic ammonium salt. 5. The use according to claim 4, wherein the inorganic ammonium salt comprises at least one of ammonium sulfate, ammonium hydrogen sulfate, ammonium chloride, ammonium nitrate, ammonium carbonate, and ammonium hydrogen carbonate. 6. purposes as claimed in claim 3 is characterized in that, when ammonium salt is used as cellulase fermentation regulator, when fermentation medium is solid medium, the addition of ammonium salt is 2.5%- 3.5%; when the fermentation medium is a liquid medium, the addition amount of ammonium salt is 1%-3% of the weight of the liquid medium.