CN111690544B - Oyster mushroom capable of producing laccase and application thereof - Google Patents

Abstract

The invention discloses a oyster mushroom strain for producing laccase and application thereof. The invention screens an Agrocybe aegerita (Agrocybe peddidae) strain Ap-SWS2 capable of secreting laccase for the first time, the strain is stored in China general microbiological culture Collection center (CGMCC) at 27 days 6 months in 2020, and the storage number is CGMCC NO. 20209. The strain fermentation liquor has high activity of the varnish enzyme, strong temperature stability and better degradation capability to lignin, and is beneficial to the application in the fields of crop straw treatment and the like.

Description

Oyster mushroom capable of producing laccase and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Agrocybe praecox strain for producing laccase and application thereof.

Background

The straw resources in China are rich, but most of the waste straw can only be incinerated because the production period of the straw is concentrated and the yield is high, so that the straw can not be effectively utilized. At present, the burning problem of the waste straws is serious, which causes a great deal of resource waste and environmental pollution. Therefore, straw upgrading applications are critical for straw treatment. Utilization and degradation of lignin in straw is difficult due to its presence, however, certain microorganisms rely on lignin-degrading enzymes to degrade lignin in straw. Although the biological method for treating straws has become a trend of the straw biological utilization research at present, microbial strains for degrading lignin generally have the problems of low enzyme activity of degrading enzyme, unsatisfactory strain degradation rate, lack of related engineering research and the like. Therefore, the development of a new straw lignin degrading high-yield strain is very important.

Lignin cannot enter cells due to its too large molecular weight, can only be degraded extracellularly, and is degraded by means of a complex peroxidase system, including manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac). Laccase (Laccases) (EC 1.10.3.2) is widely found in nature, is a polyphenol oxidase that binds multiple copper ions and oxidizes hydroquinone into p-benzoquinone under aerobic conditions. When the peroxidase hydrolyzes lignin, an electron from a benzene ring is firstly obtained to form phenoxy free radicals, and after a series of reactions, other types of free radicals are generated to destroy chemical bonds in lignin molecules, so that the lignin is degraded.

The Agrocybe praecox belongs to Agrocybe of the family of the coproallaceae (boldiiceae), is mainly distributed in reed wetlands and other areas, has few related researches at home and abroad, and has special medicinal values of tumor inhibition and the like. To date, no laccase production by Agrocybe praecox has been reported.

Disclosure of Invention

The invention aims to provide a strain of oyster mushroom Ap-SWS2 for producing laccase aiming at the defects of the prior art. The strain fermentation liquor has high activity of the varnish enzyme, strong temperature stability and better degradation capability to lignin, and is beneficial to the application in the fields of crop straw treatment and the like.

In order to achieve the aim, the invention firstly provides an Agrocybe aegerita (Agrocybe pedigrees) strain Ap-SWS 2. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 Xilu-1 of Beijing, Chaoyang, China academy of sciences microbiological research institute) at 23.6.23.2020, with the preservation number of CGMCC NO. 20209. The Ap-SWS2 strain is obtained by enriching and screening soil in the mountainous area of south of Shandong Jinan in 8 months in 2019, can secrete laccase, and can be used for treating lignin of crop straws.

The Ap-SWS2 strain (CGMCC NO.20209) obtained by screening has the following physiological and biochemical characteristics: the colony in the solid plate is loose, the surface is white flocculent, then becomes yellow brown, has brown spores, is dry, and hypha is not easy to pick up. The strain in the liquid culture medium is flocculent round-ball-shaped, yellow brown, and then connected into a sheet, and floats on the upper layer of the culture medium. Can utilize crop straws to degrade cellulose, and cannot tolerate high-concentration NaCl (figure 1).

The ITS rRNA sequence of the Ap-SWS2 strain is shown in SEQ ID No. 1. The strain was further demonstrated to be affiliated with Agrocybe praecox by blastn alignment and phylogenetic tree analysis (FIG. 2).

The invention also provides a method for producing laccase by fermenting the pleurotus ostreatus Ap-SWS2, which comprises the following steps: adding the fungus blocks in the solid plate into a seed culture medium, and performing shake culture at 25-30 ℃ for 90-100h to obtain a seed solution; taking seed liquid, adding the seed liquid into a fermentation culture medium according to an inoculation ratio of 4-6% by volume for fermentation culture at 25-30 ℃ for 100-150h, wherein the laccase activity in the fermentation liquid supernatant is higher.

The seed culture medium comprises the following components in percentage by weight: 1% of yeast extract powder, 1.5% of glucose, (NH)₄)₂SO₄0.15％，MgSO₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，pH 5.5-6.0。

The formula of the fermentation medium comprises the following components in percentage by weight: MgSO (MgSO)₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，(NH₄)₂SO₄ 0.15％，CuSO₄·5H₂O 0.00125％，MnSO₄·H₂0.0034 percent of O, 4 percent of straw and 5.0 of pH. The straws comprise straws of crops such as wheat, corn, rice and the like.

The culture conditions of the seed liquid culture are preferably as follows: the temperature is 28 ℃, the shaking culture is carried out for 96h, and the rotating speed is 180 r/min.

The culture conditions of the fermentation culture are preferably as follows: the temperature is 28 ℃, the shaking culture is carried out for 120h, and the rotating speed is 180 r/min.

The invention also provides a method for measuring the activity of the laccase on the fermentation broth of the agrocybe praecox Ap-SWS2, which is characterized in that ABTS (2, 2' -biazonitrogen-bis-3-ethylbenzthiazoline-6-sulfonic acid) is used as a substrate and reacts with the fermentation broth supernatant to measure the change rate of the light absorption value at 420 nm.

The invention also provides an application of the Agrocybe praecox Ap-SWS2 strain and a fermentation supernatant (or laccase) thereof in the degradation of crop straws (wheat, corn, rice and the like). Moreover, the Agrocybe praecox Ap-SWS2 strain and its fermentation supernatant (laccase) can also be applied in the fields of lignin degradation, environmental bioremediation, sewage treatment, printing and dyeing wastewater treatment, textile industry and the like.

The Ap-SWS2 strain can play a role in degrading straws and is a fermentation extracellular metabolite which is produced in a non-induced manner.

The invention has the following beneficial effects:

(1) the invention screens a pleurotus ostreatus strain capable of secreting laccase for the first time. Experiments prove that the strain fermentation supernatant has higher laccase activity (up to 21.7U/mL), has certain degradation capability on various straw lignins, has the lignin degradation rate on rice straws up to 26.7 percent, and can be applied to the fields of environmental bioremediation, sewage treatment, printing and dyeing wastewater treatment, textile industry and the like.

(2) The invention increases laccase family members, enriches the diversity of laccase and provides theoretical and technical support for further application of laccase in industrial large-scale production. Experiments prove that the laccase has strong temperature stability (the enzyme activity is highest at 50 ℃ and is stable at the temperature lower than 50 ℃), and strong stability under weak acid and neutral conditions (the laccase has pH4.6 (Na)₂HPO₄And-citric acid buffer solution), the enzyme activity is highest, the pH is relatively stable between 4 and 8.6, and the enzyme activity of more than 50 percent can be maintained), and the laccase has the advantages of high enzyme activity, high lignin degradation rate and the like, so the laccase has a good application prospect.

Drawings

FIG. 1 shows the growth of Ap-SWS2 strain selected according to the present invention on a medium plate;

FIG. 2 shows the evolutionary tree of Ap-SWS2 strain selected by the present invention;

FIG. 3 is a temperature optimum curve for laccase activity;

FIG. 4 is a temperature stability curve of laccase activity;

FIG. 5 is a pH optimum curve for laccase activity;

FIG. 6 is a pH stability curve of laccase activity;

FIG. 7 is a bar graph of the Ap-SWS2 strain on straw lignin degradation.

Detailed Description

The present invention is further illustrated by the following specific examples, which are provided to illustrate the present invention but are not intended to limit the scope of the present invention. In the following examples, the methods used, unless otherwise specified, are conventional in the art, and the reagents used, unless otherwise specified, are available from conventional routes. For example, LA Taq enzyme is available from Taraka and ABTS (2, 2' -diaza-bis-3-ethylbenzothiazoline-6-sulfonic acid) is available from Solebao.

Example 1 screening and identification of laccase-producing Strain Ap-SWS2 of the invention

(1) Screening of strains:

enrichment culture: collecting soil and weed samples from mountain areas in south of Shandong Jinnan in 2019 in 8 months, screening by using guaiacol as a screening agent, preparing 100mL of laccase strain screening culture medium in a 500mL conical flask, putting the samples in a flask according to the proportion of 1%, and performing shake culture at 28 ℃ and 180r/min for 96 hours.

Primary screening: adding 100 μ L of the enriched sample into an EP tube containing 900 μ L of sterile physiological saline, mixing, taking out 100 μ L of the enriched sample, adding into another EP tube containing 900 μ L of sterile physiological saline, sequentially diluting the bacterial liquid to 10¹、10²、10³、10⁴And 10⁵10 will be⁴And 10⁵And coating three parallel laccase screening plates with the diluted bacterium liquid, and culturing for 96h in an incubator at 28 ℃.

Re-screening: and selecting the strain which can grow in the plate and is red, performing liquid culture again, putting a 500mL conical flask into 100mL laccase strain screening culture medium, and performing shake flask culture at 28 ℃ and 180r/min for 96 h. And (3) measuring the degradation capability of the fermentation supernatant on ABTS, and determining the strain with the strongest degradation capability for storage. The results of laccase activity of the different strains are shown in Table 1.

The laccase strain screening culture medium formula comprises: KH (Perkin Elmer)₂PO₄：0.25g/L；NH₄NO₃：1.0g/L；CaCl₂：1.0g/L；MgSO₄：0.25g/L；FeSO₄: 1.0 mg/L; guaiacol0.4mL/L。

Table 1: laccase activity and characteristics of different strains obtained by screening

(2) Morphological identification and physicochemical characteristic identification

The physiological and biochemical characteristics of the screened strain No. 4 are as follows: the colony in the solid plate is loose, the surface is white flocculent, then becomes yellow brown, has brown spores, is dry, and hypha is not easy to pick up. The strain in the liquid culture medium is flocculent round-ball-shaped, yellow brown, and then connected into a sheet, and floats on the upper layer of the culture medium. Can utilize crop straws to degrade cellulose, and cannot tolerate high-concentration NaCl (figure 1).

(3) Molecular identification

Molecular identification of the strains: extracting genome DNA of the strain, using the genome DNA as a template for ITS rRNA amplification, and using primers as follows:

F：5’-TCCGTAGGTGAACCTGCGG-3’(SEQ ID No.2)

R：5’-TCCTCCGCTTATTGATATGC-3’(SEQ ID No.3)

PCR was performed using the synthesized primers. The PCR reaction system is as follows:

the PCR conditions were as follows:

the obtained PCR product was recovered using a gel recovery kit and sent to the EnxElite for ITS rRNA sequencing. The ITS rRNA sequence of the strain is shown in SEQ ID No. 1.

The blast comparison search shows that the target strain has higher homology with the sequence of the agrocybe praecox in Genebank, and the similarity is 99%. Meanwhile, in combination with the phylogenetic tree analysis (FIG. 2), it was further confirmed that the strain is subject to Agrocybe praecox (Agrocybe pedigrees) and named Ap-SWS 2. The strain has been preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 Xilu-1 of Beijing, Chaoyang, China academy of sciences microbiological research institute) at 23.6.23.2020, with the preservation number of CGMCC NO. 20209.

Example 2 laccase production culture of Ap-SWS2 Strain of the invention

(1) The culture medium used

Seed culture medium: 1% of yeast extract powder, 1.5% of glucose, (NH)₄)₂SO₄ 0.15％，MgSO₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，pH 5.5-6.0。

Fermentation medium 1: MgSO (MgSO)₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，(NH₄)₂SO₄ 0.15％，CuSO₄·5H₂O 0.00125％，MnSO₄·H₂0.0034 percent of O, 4 percent of rice straw and 5.0 percent of pH.

Fermentation medium 2: MgSO (MgSO)₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，(NH₄)₂SO₄ 0.15％，CuSO₄·5H₂O 0.00125％，MnSO₄·H₂0.0034 percent of O, 4 percent of corn straw and 5.0 of pH.

Fermentation medium 3: MgSO (MgSO)₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，(NH₄)₂SO₄ 0.15％，CuSO₄·5H₂O 0.00125％，MnSO₄·H₂0.0034 percent of O and 4 percent of wheat straw，pH 5.0。

(2) Culture conditions

Seed liquid culture: adding the fungus blocks in the solid flat plate into a seed culture medium for shake flask culture; the culture conditions were: the temperature is 28 ℃, shaking culture is carried out for 96h, and the rotating speed is 180r/min, thus obtaining the seed liquid.

Fermentation culture: 5mL of the seed solution was taken and added to a 250mL Erlenmeyer flask containing 100mL of the fermentation medium to conduct the culture. The fermentation temperature is 28 ℃, the fermentation time is 120h, and the rotating speed is 180r/min, so that the fermentation broth is obtained.

And (3) determining laccase activity: ABTS as substrate, pH4.6 (Na) at 50 ℃₂HPO₄Citric acid buffer solution), and the change rate of the absorbance at 420nm was measured by reacting with the supernatant of the fermentation broth (fermentation medium 1). The laccase activity was calculated to be 21.7U/mL. The supernatant protein content of the fermentation broth was determined to be 2.3mg/mL using Coomassie Brilliant blue kit.

The enzyme activity and the protein content of the fermentation broth supernatants of the fermentation culture media 2 and 3 are measured by adopting the same measuring conditions, and the results show that the laccase activity is 10.3U/mL and 9.3U/mL respectively, and the protein content is 1.02mg/mL and 0.93mg/mL respectively.

Example 3 investigation of enzymatic Properties of laccase secretion by Ap-SWS2 Strain of the present invention

(1) Optimum temperature and temperature stability of laccase

The enzyme activity is measured at different temperatures (20, 30, 40, 50, 60 and 70 ℃), the highest activity group is set as 100%, and a curve of temperature and relative enzyme activity is obtained. The results show (FIG. 3) that the optimal temperature of laccase in the crude enzyme solution is 50 ℃.

And (3) placing the enzyme solution at different temperatures (0, 10, 20, 30, 40, 50 and 60 ℃) for heat preservation for 1h, measuring the enzyme activity at 50 ℃, and obtaining a curve of temperature stability and relative enzyme activity, wherein the enzyme activity of the untreated enzyme solution is 100%. The results show (FIG. 4) that the laccase in the crude enzyme solution is relatively stable at the temperature lower than 50 ℃; after temperatures above 50 ℃, the stability drops rapidly.

(2) pH optimum and pH stability of laccase

Using buffers (50mmol/L Na) of different pH₂HPO₄-citric acidA buffer solution with a pH of 3.0-7.0; 50mmol/L Na₂HPO₄-NaH₂PO₄A buffer solution with pH of 6.6-8.0; 50mmol/L glycine-NaOH buffer solution, pH 8.6-9.6) to prepare a substrate, and respectively measuring the enzyme activity of the laccase. The results (see FIG. 5) show that laccase is at pH4.6 (Na)₂HPO₄Citrate buffer) the enzyme activity is highest.

The enzyme solution was placed in buffers (50mmol/L Na) of different pH₂HPO₄-a citric acid buffer, pH 3.0-7.0; 50mmol/L Na₂HPO₄-NaH₂PO₄A buffer solution with pH of 6.6-8.0; 50mmol/L glycine-NaOH buffer solution, pH 8.6-9.6) for 24h, and determining the corresponding activity of the laccase under the optimal condition, wherein the enzyme activity with the highest activity is 100%, so as to obtain a curve of pH stability and relative enzyme activity. The result (shown in figure 5) shows that the laccase is relatively stable between pH 4-8.6 and can keep more than 50% of enzyme activity.

Example 4 degradation result of the Ap-SWS2 strain of the invention on straw lignin

During strain culture, different crop straws (rice, wheat and corn, shown as fermentation culture media 1-3 respectively) are added into a fermentation culture medium, and lignin contents in different culture times (0h, 24h, 72h and 120h) are measured.

The results show (FIG. 7) that the Ap-SWS2 strain can degrade lignin of various crop straws. The degradation effect on the rice lignin is most obvious, after the rice lignin is cultured for 120 hours, the lignin content is reduced to 16.2% from the original 22.1%, and the degradation rate can reach 26.7%. The lignin in the wheat and corn straws has certain degradation capability, and the degradation rates are 22.9 percent and 16.9 percent respectively.

SEQUENCE LISTING

<110> institute of biological research of academy of sciences of Shandong province

<120> oyster mushroom for producing laccase and application thereof

<130> 0

<160> 3

<170> PatentIn version 3.3

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<211> 688

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<213> ITS rRNA sequence of Agrocybe praecox (Agrocybe pedigrees) strain Ap-SWS2

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tccctccgcc ttattgatat gcttaagttc agcgggtagt cctacctgat ttgaggtcaa 60

attgtcattt gtgttgtccc gagagacggt tagaagcagc gcaaacccat tatagcagac 120

gtccacggcg tagataatta tcacaccaat agacggttcc actgcggggc accggctaat 180

acatttaagg ggagcagact aatgaaagcc agcaaaaaga cccccaagtc caagccatta 240

tcagcaaaag ctaataaggt tgagaattta atgacactca aacaggcatg ctcctcggaa 300

taccaaggag cgcaaggtgc gttcaaagat tcgatgattc actgaattct gcaattcaca 360

ttacttatcg catttcgctg cgttcttcat cgatgcgaga gccaagagat ccgttgctga 420

aagttgtata tagtttatag gcactaggcc atatacaata cattctgtta cattctttgg 480

ggtatgtgta agacataggc ctggataata caaggaaagc cgactcgtga aagccagcag 540

tcctcccgac cgagttgcct cggaaagtta tcgtccaggt ctacaaaggg tgcacaggtg 600

gagatataaa gatgacgggc gagcacatgt ccccgagagg accagctaca accacgccag 660

gtttattcaa taatgatcct tccgcagg 688

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<223> primer F

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tccgtaggtg aacctgcgg 19

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tcctccgctt attgatatgc 20

Claims (4)

1. Oyster mushroom (oyster mushroom) for producing laccaseAgrocybe pediades) The strain is Ap-SWS2, and the preservation number of the strain is CGMCC NO. 20209.

2. The use of the strain of Agrocybe praecox as claimed in claim 1 for producing laccase by fermentation and degrading lignin.

3. The method for producing laccase by fermentation using the Agrocybe praecox strain of claim 1, wherein the strain is a strain that is capable of producing laccase,

adding the fungus blocks in the solid plate into a seed culture medium, and performing shake culture at 25-30 ℃ for 90-100h to obtain a seed solution; taking seed liquid, adding the seed liquid into a fermentation culture medium according to an inoculation ratio of 4-6% by volume for fermentation culture at 25-30 ℃ for 100-150h to obtain fermentation liquid supernatant containing laccase;

the seed culture medium is as follows: 1% of yeast extract powder, 1.5% of glucose, (NH)₄)₂SO₄ 0.15％，MgSO₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，pH 5.5-6.0；

The fermentation medium is as follows: MgSO (MgSO)₄·7H₂O 0.05％，KH₂PO₄ 0.08％，K₂HPO₄·3H₂O 0.02％，(NH₄) ₂SO₄ 0.15％，CuSO₄·5H₂O 0.00125％，MnSO₄·H₂0.0034 percent of O, 4 percent of straw and 5.0 of pH.

4. The method for producing laccase by fermentation according to claim 3, wherein the straw is wheat straw, corn straw or rice straw.

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