CN114164188B - Method for producing laccase by degrading reed stalks with yak rumen anaerobic fungus and methane bacteria co-culture and application - Google Patents

Abstract

The invention relates to the field of biotechnology renewable energy, in particular to a method for producing laccase by degrading reed stalks with a co-culture of anaerobic fungi and methane bacteria naturally symbiotic with yak rumen and an application thereof, wherein the co-culture is YakQH5, consists of anaerobic fungi (Neocallimastix frontalis) and methane bacteria (Methanobrevibacter gottschuki), is preserved in China general microbiological culture Collection center (CGMCC) 3, 9 and 2020, and has a preservation number of CGMCC No. 19299. The mixed culture YakQH5 can degrade reed stalks and produce a large amount of laccase, the laccase activity is as high as 60.5U/mL, and the industrial application value is important.

Description

Method for producing laccase by degrading reed stalks with yak rumen anaerobic fungus and methane bacteria co-culture and application

Technical Field

The invention relates to the field of biotechnology renewable energy, in particular to a method for producing laccase by degrading reed stalks with a co-culture of yak rumen anaerobic fungi and methane bacteria and application thereof.

Background

Traditional energy substances face the danger of exhaustion, and when the traditional energy substances are used, a large amount of greenhouse gases are generated, so that the environment is polluted. The relief of the three crises of 'food, energy and environment' which are coming to people at present is one of the important ways to realize the strategy of agricultural sustainable development. The roughage is feed with natural water content below 60% and crude fiber content above 18% and fed in air-dried form, including hay, crop straw, leaves, pericarp, distiller's grains, blighted shells, etc. The coarse fodder has high fiber content, low protein and mineral content, and poor palatability. The main components of coarse fodder such as straw, hay and chaff are lignocellulose, including lignin, cellulose and hemicellulose. Straw, hay, blighted shells and the like are beneficial to use and harmful to abandon, and can bring environmental pollution and other harmful places as wastes, and can be changed into valuables by fully utilizing the straws, the hay, the blighted shells and the like as important renewable resources, thereby benefiting mankind.

Lignin and hemicellulose in roughage form a natural barrier in the form of covalent bonds, which entraps cellulose therein and prevents the cellulose from being enzymatically degraded. The cellulose, hemicellulose and lignin in the lignocellulose form a compact structure together. Lignin is a phenolic polymer formed by 5-hydroxy coniferyl alcohol, p-coumaryl alcohol, sinapyl alcohol and coniferyl alcohol, which are tightly linked together in the form of covalent bonds and are the core skeleton for stabilizing cellulose and hemicellulose. Lignin is chemically stable, resulting in difficult degradation in the traditional manner. The pretreatment of the roughage comprises physical, chemical and biological methods, and the roughage such as straws, hay, chaff and the like degraded by microorganisms capable of secreting effective enzymes at present has the characteristics of low energy consumption, low cost, safe operation and no pollution, thereby causing more and more extensive attention at home and abroad.

Biodegradation of lignin is of great importance in environmental and energy sciences. Microbial degradation of lignocellulose is due to the co-action of a series of enzymes secreted by it. In the degradation process, enzymes such as xylanase, carboxymethyl cellulose and laccase cooperate with each other to play an important role in the reaction process of degrading lignocellulose. Laccase is an important member of a lignin degradation enzyme system, is a key enzyme in the lignin degradation process, realizes the degradation of lignin by attacking phenolic compounds in the lignin, and usually extracts electrons from the lignin to reduce oxygen in the air into water.

Laccases were first discovered and isolated in sumac in japan in 1883, and studies on laccases have been continued for over 140 years to date. Laccases are widely distributed in nature in higher plants, microorganisms, algae, insects, and invertebrates, and many organisms can produce laccases, the main sources of which are bacteria and various aerobic fungi in soil. Laccase is a copper-rich oxidase capable of oxidizing various phenolic and non-phenolic compounds, belongs to copper blue oxidase, takes molecular oxygen as a final electron acceptor, and can catalyze polymerization, degradation and conversion of lignin and a large class of phenolic and arylamine substances. The peptide chain of laccase is mainly composed of about 500 amino acids, and the sugar aglycone accounts for 10% -45% of the whole molecule. Copper ions participate in the construction of laccase active centers, jointly form the most important part in laccase structure, determine the activity and specificity of laccase, and are areas for combining substrates and directly participating in bond cleavage formation. Because the laccase has wide substrate and non-substrate specificity, the product generated by catalysis does not pollute the environment, and the byproduct of catalytic reaction is only water, the laccase has huge application potential in various fields as a green biocatalyst, and especially plays an important role in the aspects of food industry, paper industry, textile industry, medicine, synthetic chemistry, cosmetics, soil bioremediation, biodegradation, biofuel cells, environmental protection and the like. The laccase as a biological enzyme can degrade pollutants which are difficult to degrade, such as phenols, amines, carboxylic acids and the like, into small molecular substances with weaker environmental toxicity under mild conditions, and has huge application potential and wide market prospect. At present, the laccase research mainly comprises the laccase secreted by white rot fungi in basidiomycota, and the Trichoderma reesei is also an important production strain of industrial laccase.

The superior properties of laccases in all aspects have led to an increasing interest. Although the research on laccase has been greatly advanced, and the biochemical research, the three-dimensional structure, the catalytic mechanism and the like are further researched, the research on the practical application of laccase is superficial, the difference of a laccase determination method and a determination system is large, the comparison between enzyme activity and yield is difficult, the reports on large-scale industrial production of laccase are few, the synthesis cost of laccase is high, the production level is low and the like, so that the screening of excellent high-yield laccase strains, the obtaining of laccase with stable performance and the realization of high-efficiency production of laccase are urgent.

Anaerobic fungi play an important role in the degradation of lignocellulose. Most species in anaerobic fungi can secrete high-activity enzymes including cellulase, hemicellulase, esterase, laccase and the like through rhizoid, and the enzymes synergistically decompose and utilize substances with complex and crystalline structures, such as cellulose, hemicellulose, pectin, lignin and the like. Some methanogens can use metabolites of anaerobic fungi, such as hydrogen, methane, formic acid, etc., to form stable co-cultures with anaerobic fungi, i.e.: the process promotes the growth of anaerobic fungi and methanobacteria, and simultaneously obviously improves the activity of various lignocellulose degrading enzymes generated by degrading lignocellulose by the anaerobic fungi and the degradation capability of the lignocellulose.

The yak adapts to the severe cold ecological condition, and is resistant to rough feeding, severe cold and hypoxia. The rumen of the yak inhabits uniquely, complexly and variously, and a large number of microbial communities synergistically degrade the wild pasture efficiently to provide survival energy and nutrient substances for the yak. The invention unexpectedly discovers that the laccase enzyme activity of degrading reed stalks by adopting the anaerobic fungus and methane bacteria co-culture YakQH5 separated from the rumen of grazing yaks in Hainan, Qinghai province can be as high as 60.5U/mL, and is far higher than that of degrading wastes such as other stalks (wheat stalks, rice stalks, corn stalks, sorghum stalks, oat stalks, soybean stalks, cotton stalks, flax stalks, sesame stalks, soybean stalks, potato stalks, tomato residues, wheat husks, rice husks, melon seed husks, walnut husks, peanut husks, coconut shells, bran, apple husks, soybean husks, banana husks, corn husks, sisal hemp, wood chips and Chinese wildrye), and an unexpected effect is obtained.

Disclosure of Invention

In view of the above technical problems, the present invention provides a method for producing laccase by degrading waste materials with a naturally symbiotic mixed culture consisting of anaerobic fungi (Neocallimastix frontalis) and methanobacteria (Methanobacter gottschalkii), the method comprising the steps of:

(1) preparing a mixed culture microbial inoculum: inoculating the mixed culture into an anaerobic culture medium taking wheat straws as a substrate, adding a compound antibiotic, and carrying out anaerobic culture to obtain a high-activity microbial inoculum;

(2) producing laccase: and (2) absorbing the microbial inoculum obtained in the step (1), inoculating the microbial inoculum into an anaerobic culture medium taking reed stalks as a substrate, adding a compound antibiotic, and carrying out anaerobic culture to obtain the microbial inoculum.

Preferably, the mixed culture is YakQH5 (mixed culture of Neocallimastix frontalis and Methanobacter gottschalkii), which is preserved in China general microbiological culture Collection center (CGMCC) at 3-9 th of 2020 with the preservation number of CGMCC No.19299 and the preservation address of: the microbial research institute of the institute of science and technology of china, No. 3, west way, No.1, north chen, chaoyang district, telephone: 010-64807355. The ITS sequence of Neocallimastix frontalis in the mixed culture YakQH5 is shown as SEQ ID NO.1, and the 16S rDNA sequence of Methanobacter gottschalkii is shown as SEQ ID NO. 2.

Preferably, the inoculum size of the mixed culture in step (1) is 10% v/v.

Preferably, the inoculation amount of the microbial inoculum in the step (2) is 10% v/v.

Preferably, the content of the reed stalks in the step (2) is 1% w/v.

Preferably, the temperature of the anaerobic culture in the step (1) is 39 ℃ and the time is 72 h.

Preferably, the temperature of the anaerobic culture in the step (2) is 39 ℃ and the time is 120 h.

Preferably, the antibiotic complex consists of 1600IU/mL penicillin sodium and 2000IU/mL streptomycin sulfate.

Preferably, the complex antibiotic is added in an amount of 1% v/v.

Preferably, the anaerobic fungus culture medium has the formula as follows: yeast extract 1.0g, NaHCO ₃ 7.0g, 1.0g/L resazurin 1mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II 16.5mL, distilled water constant volume to 1000 mL; the preparation steps of the salt solution I are as follows: NaCl 6g, (NH) ₄ ) ₂ SO ₄ 3g，KH ₂ PO ₄ 3g，CaCl ₂ ·2H ₂ O 0.4g，MgSO ₄ ·2H ₂ 0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: 4g K ₂ HPO ₄ And distilled water is added to the volume of 1000 mL.

Preferably, in the step (2), the reed stems are inoculated, oxygen is removed, carbon dioxide is filled, and high-temperature and high-pressure sterilization is carried out.

The beneficial effects of the invention are: according to the invention, a natural symbiotic mixed culture YakQH5 co-culture of Neocallimastix frontalis and Methanobrevibacter gottschalkii is adopted to carry out anaerobic fermentation to degrade reed stalks, the laccase degrading enzyme can be used for producing laccase, the activity of the produced laccase reaches the maximum value of 60.5U/mL, and is obviously higher than that of the laccase produced by degrading other substrates (wheat straws, rice straws, corn straws, sorghum straws, oat straws, soybean straws, cotton straws, flax straws, sesame straws, soybean straws, potato straws, tomato pomace, wheat husks, rice husks, melon seed shells, walnut shells, peanut shells, coconut shells, wheat bran, apple husks, soybean husks, banana husks, corn husks, sisal, wood chips and leymus chinensis), and the laccase degrading enzyme has obvious progress; the co-culture adopted in the invention can survive and pass in vitro through preservation, is convenient for popularization and provides great convenience for production.

Detailed Description

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are all obtained from commercial sources unless otherwise specified; the culture medium is a formula which is improved and simplified by the inventor of the patent on the conventional anaerobic fungus culture medium, and comprises the following components:

the anaerobic fungus culture medium comprises the following components in percentage by weight: yeast extract 1.0g, NaHCO ₃ 7.0g of resazurin (1.0g/L)1mL, L-cysteine hydrochloride 1.7g, salt solution I82.5 mL, salt solution II 16.5mL, and distilled water with constant volume of 1000 mL; the salt solution I comprises 6g of NaCl, (NH) ₄ ) ₂ SO ₄ 3g，KH ₂ PO ₄ 3g，CaCl ₂ ·2H ₂ O 0.4g，MgSO ₄ ·2H ₂ 0.6g of O and distilled water with constant volume of 1000 mL; the salt solution II comprises 4gK ₂ HPO ₄ And distilled water is added to the volume of 1000 mL.

Example 1 preparation of symbiotic culture YakQH5 bacterial agent

Sucking 1mL of mixed culture YakQH5 of isocollimatix frontalis and Methanobacter gottschalkii, inoculating the mixed culture YakQH5 into a 20mL Henry anaerobic tube containing 9mL of anaerobic culture medium taking air-dried and crushed wheat straws as a substrate, simultaneously adding 0.1mL of compound antibiotics (penicillin and 2000IU/mL streptomycin sulfate at a final concentration), and carrying out anaerobic culture at 39 ℃ for 72h to reach a growth peak, wherein the fermentation liquid is a high-activity microbial inoculum.

Example 2 Co-culture YakQH5 degradation of waste to produce laccase

45mL of liquid basic culture medium is contained in a 100mL anaerobic fermentation bottle, and 0.5g of dried and crushed wheat straw, rice straw, corn straw, sorghum straw, oat straw, soybean straw, cotton straw, flax straw, sesame straw, soybean straw, potato straw, tomato pomace, wheat hull, rice hull, melon seed hull, walnut hull, peanut hull, coconut hull, bran, apple hull, soybean hull, banana hull, corn hull, sisal, wood dust, leymus and reed straw are respectively used as substrates. Deoxygenated and then autoclaved. 5mL of the mixed culture YakQH5 after 72 hours of subculture was inoculated into the above anaerobic medium to which 27 waste substrates were added, respectively, by aspiration with a sterile syringe, and 0.5mL of a complex antibiotic (penicillin and streptomycin sulfate at a final concentration of 1600IU/mL and 2000 IU/mL) was added, followed by anaerobic culture at 39 ℃ for 5 days, to thereby set up 3 parallel experiments.

Example 3 laccase content determination

The laccase activity of the fermentation broth in the anaerobic flask of example 2 was determined at 24h intervals. Laccase activity determination method (using ABTS method):

(1) centrifuging the fermentation liquor at 4 deg.C and 5000r/min for 15min, and collecting supernatant as crude enzyme solution;

(2) preparing a 0.5mmol/LABTS solution: a first reagent: 0.0384g ABTS constant volume is 10 mL; and a second reagent: 0.0134g of potassium persulfate is metered to 10mL, and the volume of the first reagent and the second reagent is 1: 1, mixing, and obtaining 7mmol/L ABTS solution after 12 hours of light shielding, and obtaining 0.5mol/LABTS solution after dilution by 14 times;

(3) absorbing 1.00mL of crude enzyme solution, adding 2.00mL of acetic acid-sodium acetate buffer solution (pH6.8) to dilute for 3 times, bathing the diluted enzyme solution in water at 39 ℃ for 30min, taking 2.9mL of ABTS solution during measurement, adding 0.1mL of preheated diluted enzyme solution, quickly placing the solution into an ultraviolet spectrophotometer, and recording the absorbance value from one point (A) ₁ ) Change to another point (A) ₂ ) Time t according to enzyme activityCalculating enzyme activity:

A ₁ 、A ₂ two end values of the change of absorbance, t is the absorbance value from A ₁ Increase to A ₂ Elapsed time(s); enzyme activity unit is defined as the amount of enzyme required to oxidize 1 μmol of substrate per minute is 1 enzyme activity unit (U); the unit of the enzyme activity is U/mL.

The analysis of 27 waste components is shown in Table 1, and the measurement results of laccase activity are shown in Table 2.

Table 127 lignocellulosic compositions of waste substrates

TABLE 2 laccase activity of the co-culture YakQH5 produced by degrading 27 wastes respectively in 5-day culture period

Note: ^a and b, c, d represent significance of difference (p)<0.05)

The above experimental results show that: in the 5-day culture period, the activity of the symbiotic culture YakQH5 of Neocallimastix frontalis and Methanobacter gottschalkii for degrading 27 wastes to generate extracellular enzyme-laccase respectively reaches the highest value: 11.0U/mL wheat straw, 8.7U/mL rice straw, 9.0U/mL corn straw, 8.3U/mL sorghum straw, 10.6U/mL oat straw, 7.0U/mL soybean straw, 6.5U/mL cotton straw, 15.0U/mL flax straw, 6.1U/mL sesame straw, 11.5U/mL soybean straw, 7.1U/mL potato straw, 30.0U/mL tomato pomace, 9.2U/mL wheat hull, 1.1U/mL rice hull, 0 melon seed hull, 0 walnut hull, 0 peanut hull, 1.8U/mL coconut hull, 9.3U/mL bran, 2.9U/mL apple hull, 15.0U/mL soybean hull, 2.0U/mL banana hull, 25.0U/mL corn hull, 33.8U/mL sheep grass, 6.4U/mL hemp straw, 1.9U/mL sisal wood chip, and 1.9U/mL wood chip, The activity of laccase generated by degrading reed stalks is as high as 60.5U/mL, is obviously higher than that of laccase generated by degrading other substrates, and has important industrial application value.

Sequence listing

<110> institute of biological research of science institute of Gansu province

Method for producing laccase by degrading reed stalks with yak rumen anaerobic fungus and methane bacteria co-culture and application

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<170> SIPOSequenceListing 1.0

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ggaaggatca ttaataaatg gctccggttt ttttaaaatg gagcacttaa taatccctac 180

cctttgtgaa tttgttgttt gaaataattt tttttaaaaa aaaattattg aattgtcggg 240

ccctgtttaa ttggaatcct tttcggacgt gaaaattccc aaggggataa ggcaattcaa 300

taaatttttt taaaaaagac ctttttttta aattaaactt tttgtattca ttttgtctaa 360

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aaaatattct cataaaaaat attttgtttt tatggttttg agtgttttac aacggataca 600

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atagggcaca ctaaggggac cgcctatgat aaataggagg aaggagtgga cgacggtagg 1020

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cgacaccgaa aggtggaggt aatcctctaa acttagtcgt agttcggatt gaggactgta 1140

actcgttctc atgaagctgg aatgtgtag 1169

Claims (9)

1. A method for producing laccase by degrading waste by natural symbiotic mixed culture, which is characterized in that the mixed culture consists of anaerobic fungi (Neocallimastix frontalis) and methanobacteria (Methanobacter gottschalkii), and the method comprises the following steps:

(1) preparing a mixed culture microbial inoculum: inoculating the mixed culture into an anaerobic culture medium taking wheat straws as a substrate, adding a compound antibiotic, and carrying out anaerobic culture to obtain a high-activity microbial inoculum;

(2) and (3) laccase production: absorbing the microbial inoculum obtained in the step (1) to be inoculated into an anaerobic culture medium taking reed stalks as substrates, adding compound antibiotics, and carrying out anaerobic culture to obtain the microbial inoculum; the natural symbiotic mixed culture is YakQH5, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) No.19299 in 3-9 months in 2020, and has the preservation address as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, and Beijing: 010-64807355.

2. The method of claim 1, wherein the amount of mixed culture inoculated in step (1) is 10% v/v.

3. The method of claim 1, wherein the inoculum size of the inoculum in step (2) is 10% v/v.

4. The method of claim 1, wherein the reed straw content in step (2) is 1% w/v.

5. The method of claim 1, wherein the anaerobic cultivation in step (1) is carried out at a temperature of 39 ℃ for 72 hours.

6. The method of claim 1, wherein the anaerobic cultivation in the step (2) is performed at a temperature of 39 ℃ for 120 hours.

7. The method of claim 1, wherein the antibiotic cocktail consists of penicillin sodium at a final concentration of 1600IU/mL and streptomycin sulfate at a final concentration of 2000 IU/mL; the addition amount of the complex antibiotic was 1% v/v.

8. The method of claim 1, wherein the anaerobic culture medium is formulated as: yeast extract 1.0g, NaHCO ₃ 7.0g, 1.0g/L of resazurin, 1.7g of L-cysteine hydrochloride, 82.5mL of salt solution I, 16.5mL of salt solution II and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution I are as follows: NaCl 6g, (NH) ₄ ) ₂ SO ₄ 3g，KH ₂ PO ₄ 3g，CaCl ₂ ·2H ₂ O 0.4g，MgSO ₄ ·2H ₂ 0.6g of O and distilled water with constant volume of 1000 mL; the preparation steps of the salt solution II are as follows: 4g K ₂ HPO ₄ And distilled water is added to the volume of 1000 mL.

9. The method of claim 1, wherein in step (2), the reed stems are inoculated, deoxygenated, aerated with carbon dioxide, and autoclaved.