Pleurotus ostreatus, fungicide and application of pleurotus ostreatus and fungicide

Abstract

The invention provides pleurotus ostreatus, a fungicide and application of the fungicide, and belongs to the technical field of microbial preparations. The oyster mushroom provided by the invention can be used for fermentation production of laccase, the enzyme activity of the laccase can reach 480.22 + / -7.53 U / mL at most, and a theoretical basis is provided for industrial production of the laccase.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preparations, and in particular relates to a strain of Pleurotus ostreatus, a microbial preparation and application thereof. Background Art

[0002] Fungi are an important part of nature. They are widely present in nature and there are many kinds of them. Among the known fungi, there is a class of fungi that mainly live in trees or soil. They are named wood-rotting fungi because they can cause trees to rot. There are two main types of decay of wood-rotting fungi, which can be divided into oyster mushrooms and brown rot fungi. Among them, oyster mushrooms have a rich extracellular enzyme system: lignin peroxidase (LigninPeroxidase LiP), manganese peroxidase (ManganesePeroxidase MnP) and laccase (Laccase), which can react with the components in wood. It can selectively degrade lignin in wood (mainly deciduous forests) or degrade lignin, cellulose and hemicellulose at the same time, exposing white cellulose, making the wood appear white, hence the name.

[0003] Pleurotus ostreatus is not only used in the degradation of lignin, but also widely used in biopulping, food processing industry, environmental remediation, wastewater treatment, etc. Compared with lignin peroxidase and manganese peroxidase, laccase does not require other substances as initiators and auxiliary agents in the process of catalytic oxidation of substrates, so the research and application of laccase is more extensive and in-depth.

[0004] However, the current production of laccase using Pleurotus ostreatus fermentation has a low yield, most of which are between 120 and 150 U / mL, and the production cycle is long and the enzyme production process is slow. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Pleurotus ostreatus, a bacterial agent and applications thereof. The Pleurotus ostreatus of the present invention has a high yield of laccase.

[0006] The invention provides a Pleurotus ostreatus strain XH005, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Committee, and the preservation number is: CGMCC No.41421.

[0007] The present invention also provides a culture of Pleurotus ostreatus XH005 described in the above scheme.

[0008] The present invention also provides a bacterial agent, comprising the Pleurotus ostreatus XH005 described in the above scheme.

[0009] The present invention also provides the use of Pleurotus ostreatus XH005, the culture or the bacterial agent described in the above scheme in producing laccase and / or degrading lignocellulose.

[0010] The present invention also provides a method for producing laccase, comprising the following steps: inoculating the seed liquid of Pleurotus ostreatus XH005 described in the above scheme into a culture medium, culturing, and obtaining a culture containing laccase.

[0011] Preferably, the culture medium includes a first enzyme-producing culture medium and / or a second enzyme-producing culture medium; the first enzyme-producing culture medium uses water as a solvent and includes components in the following concentrations: 10-50 g / L glucose, 2-10 g / L yeast extract and 0.5-2.5 mmol / L copper sulfate; or, the second enzyme-producing culture medium uses water as a solvent and includes components in the following concentrations: 30 g / L glucose, 4 g / L peptone and 1.5 g / L magnesium sulfate.

[0012] Preferably, the concentration of glucose in the first enzyme production medium is 39.45-40 g / L.

[0013] Preferably, the concentration of yeast extract in the first enzyme production medium is 5.97-6 g / L.

[0014] Preferably, the concentration of copper sulfate in the first enzyme production medium is 0.85-1 mmol / L.

[0015] Preferably, the number of effective spores of Pleurotus ostreatus in the seed solution is 1×10 8 / mL; the inoculation amount of the seed liquid accounts for 1% to 5% of the total volume of the culture medium; and the culture time is 7 to 9 days.

[0016] The present invention provides a Pleurotus ostreatus XH005, which is characterized in that it is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee, and the deposit number is: CGMCC No. 41421. The Pleurotus ostreatus of the present invention can be used for fermentation to produce laccase, and the maximum laccase activity can reach 480.22±7.53U / mL, providing a theoretical basis for the industrial production of laccase.

[0017] Biological Deposit Description

[0018] Pleurotus sp. XH005 was deposited at the General Microbiology Center of China Culture Collection Administration on August 6, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is: CGMCCNo.41421. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 XH005 was cultured on potato dextrose agar medium;

[0021] Figure 2 for XH005 on guaiacol-potato dextrose agar medium;

[0022] Figure 3 is the ITS sequence alignment result;

[0023] Figure 4 This is the laccase production curve of XH005;

[0024] Figure 5 This is the result diagram of the effect of carbon source on laccase production of XH005;

[0025] Figure 6 This is the result diagram of the effect of glucose addition on laccase production of XH005;

[0026] Figure 7 This is the result diagram of the effect of nitrogen source on laccase production of XH005;

[0027] Figure 8 The effect of yeast extract powder addition on laccase production of XH005;

[0028] Fig. 9 This is the result diagram of the effect of inoculation amount on laccase production of XH005;

[0029] Fig.10 This is the result diagram of the effect of metal ions on the laccase production of XH005;

[0030] Fig.11 This is the result diagram of the effect of copper ion addition on laccase production of XH005;

[0031] Fig.12 This is the result diagram of the effect of pH on the laccase production of XH005;

[0032] Fig.13 The response surface of the interaction between the addition amount of glucose and yeast extract powder on the enzyme activity;

[0033] Fig.14 The response surface of the interaction between glucose addition and copper ion concentration on enzyme activity;

[0034] Fig.15The response surface of the interaction between yeast extract powder addition and inoculum size on enzyme activity;

[0035] Fig.16 The response surface of the interaction between glucose addition amount and inoculum amount on enzyme activity;

[0036] Fig.17 The response surface of the interaction between yeast extract powder addition and copper ion concentration on enzyme activity;

[0037] Fig.18 This is the response surface of the interaction between the addition amount and copper ion concentration affecting enzyme activity. DETAILED DESCRIPTION

[0038] The invention provides a Pleurotus ostreatus strain XH005, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Committee, and the preservation number is: CGMCC No.41421.

[0039] In the present invention, Pleurotus ostreatus XH005 is separated from rotten wood in Nanshan, Urumqi, Xinjiang, and can produce abundant enzymes during its growth.

[0040] The present invention also provides the culture of Pleurotus ostreatus XH005.

[0041] The present invention also provides a bacterial agent, comprising the Pleurotus ostreatus XH005 described in the above scheme.

[0042] The present invention also provides the use of Pleurotus ostreatus XH005, the culture or the bacterial agent described in the above scheme in producing laccase and / or degrading lignocellulose.

[0043] The present invention also provides a method for producing laccase, comprising the following steps:

[0044] The seed liquid of Pleurotus ostreatus XH005 described in the above scheme is inoculated into a culture medium and cultured to obtain a culture containing laccase.

[0045] In the present invention, the culture medium preferably includes an enzyme-producing culture medium; the culture medium includes a first enzyme-producing culture medium or a second enzyme-producing culture medium (basic enzyme-producing culture medium); the first enzyme-producing culture medium uses water as a solvent and includes components in the following concentrations: 10 to 50 g / L of glucose, 2 to 10 g / L of yeast extract powder and 0.5 to 2.5 mmol / L of copper sulfate; or, the second enzyme-producing culture medium uses water as a solvent and includes components in the following concentrations: 30 g / L of glucose, 4 g / L of peptone and 1.5 g / L of magnesium sulfate.

[0046] In the present invention, the pH value of the culture medium is preferably 5.

[0047] In the present invention, the concentration of glucose in the first enzyme production medium is preferably 39.45-40 g / L.

[0048] In the present invention, the concentration of yeast extract in the first enzyme production medium is preferably 5.97-6 g / L.

[0049] In the present invention, the copper sulfate in the first enzyme production culture medium is preferably CuSO4·5H2O; the concentration of the CuSO4·5H2O is preferably 0.85-1 mmol / L.

[0050] In the present invention, the number of effective spores of Pleurotus ostreatus in the seed solution is 1×10 8 The inoculation amount of the seed solution accounts for 1% to 5% of the total volume of the culture medium, preferably 3%.

[0051] In the present invention, the seed solution is preferably prepared by the following method:

[0052] The mycelium of the XH005 strain was inoculated on a PDA plate and activated at 30°C until the mycelium covered the entire plate; a bacterial cake was taken from the plate covered with mycelium, broken up, and inoculated into a PDB medium for expansion culture to obtain a seed solution.

[0053] In the present invention, the XH005 strain is stored in a PDA slant; the taking of a cake block from a plate full of hyphae preferably includes taking a cake block with a diameter of 1 cm using a sterile puncher; breaking the cake block preferably includes breaking the cake block using a hand-held tissue grinder and a sterile rotor. In the present invention, the PDB culture medium is contained in a 250 mL Erlenmeyer flask; the temperature of the expanded culture is preferably 30°C; the time of the expanded culture is preferably 4 days; the expanded culture is preferably shaker culture; the speed of the shaker is preferably 150 r / min.

[0054] In the present invention, the culture time is preferably 7 to 9 days, more preferably 8 days; the culture temperature is preferably 30°C; the culture is preferably shaker culture; and the rotation speed of the shaker culture is preferably 150 r / min.

[0055] In one embodiment of the present invention, the concentration of glucose in the enzyme production medium is 40 g / L, the concentration of yeast extract is 6 g / L, the concentration of copper sulfate is 1 mmol / L, the inoculation amount of the seed solution accounts for 3% of the total volume of the culture medium, and the culture time is preferably 8 days.

[0056] In another embodiment of the present invention, the concentration of glucose in the enzyme production medium is 39.45 g / L, the concentration of yeast extract powder is 5.97 g / L, the concentration of copper sulfate is 0.85 mmol / L, the inoculation amount of the seed solution accounts for 3% of the total volume of the culture medium, and the culture time is preferably 8 days.

[0057] The culture conditions of the above examples can significantly increase the yield of laccase produced by XH005. It can be seen that the present invention can effectively increase the yield of laccase produced by XH005 by reasonably setting the fermentation conditions of Pleurotus ostreatus XH005, providing a theoretical basis for the large-scale production of laccase.

[0058] Furthermore, the culture conditions of the present invention can improve the ability of XH005 to degrade lignocellulose, which not only provides an effective way for the biodegradation of agricultural straw waste, but also reduces environmental pollution.

[0059] In order to further illustrate the present invention, a strain of Pleurotus ostreatus, a fungal agent and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0060] The data results obtained in the embodiments of the present invention were processed and statistically analyzed using Microsoft Excel 2019 and SPSS24, and graphs were drawn using GrapHpadPism9.5 software.

[0061] Example 1 Isolation of strains

[0062] 1. XH005 was obtained from rotten wood in Nanshan, Urumqi, Xinjiang. The mycelium was cut into 0.5 cm squares and placed in the middle of the culture medium. The mycelium was inoculated on the potato dextrose agar medium containing chloramphenicol. The inoculated culture medium was placed in an incubator at 30°C and the growth was recorded every day. After it grew well in the potato dextrose agar medium containing chloramphenicol, it was inoculated on the potato dextrose agar medium and cultured at 30°C. Figure 1 shown.

[0063] 2. Identification of strains

[0064] The isolated XH005 was inoculated into guaiacol-potato dextrose agar medium to form a yellow-brown oxidation zone. The color development results of guaiacol-potato dextrose agar medium are as follows: Figure 2 shown.

[0065] 3. The fungus was sent to Shanghai Biotechnology for ITS sequence detection. The fungus species was Pleurotus ostreatus through BLAST sequence homology analysis in NCBI. Figure 3 shown.

[0066] Example 1

[0067] 1.1 Preparation of seed solution

[0068] The mycelium of the XH005 strain preserved on the PDA slope was picked up and inoculated on a new PDA plate for activation. It was cultured at 30°C until the mycelium covered the entire plate. A sterile puncher was used to prepare a 1-cm diameter cake block. Then, 3 cake blocks were taken and broken up with a handheld tissue grinder and a sterile rotor. The blocks were transferred to the PDB medium in a 250-mL Erlenmeyer flask and cultured at 30°C and 150 r / min in a shaking incubator for 4 days as the seed solution.

[0069] 1.2 Enzyme production curve of strains

[0070] The seed liquid was inoculated into the basic enzyme production medium at an inoculum of 3%, and cultured at 30°C and 150 r / min in a shaking incubator for 8 days. The laccase activity of the fermentation liquid was measured every 1 day starting from the 3rd day.

[0071] Basic enzyme production medium: glucose 30g / L, peptone 4g / L, magnesium sulfate 1.5g / L, natural pH.

[0072] Enzyme production curve Figure 4 shown.

[0073] Depend on Figure 4 It can be seen that the enzyme activity of the strain increased slowly from 3 to 4 days, and the enzyme activity increased rapidly from 4 to 8 days, reaching a maximum of 171.54±4.43U / mL on the 8th day. After the 8th day, the enzyme activity began to decline. After the 8th day, the mycelium may have aged due to the long growth time, which led to a decrease in enzyme production capacity. Therefore, the 8th day was determined as the culture time with the highest laccase activity. Based on this, the following single-factor experiments all measured the laccase activity on the 8th day to determine the optimal culture conditions.

[0074] 1.3 Single Factor Experiment

[0075] 1.3.1 Effect of carbon source type and addition amount on laccase production

[0076] The seed liquid of strain XH005 was inoculated into the basic enzyme production culture medium at an inoculum size of 3%, and 30 g / L of maltose, fructose, mannitol, soluble starch and glucose were added as carbon sources. Other conditions were kept unchanged and cultured at 30°C and 150 r / min for 8 days. The supernatant of the fermentation liquid was taken to determine the enzyme activity, the effects of different carbon sources on the enzyme production of XH005 were investigated, and the optimal carbon source was determined.

[0077] Results Figure 5 .Depend on Figure 5It can be seen that when maltose, glucose, fructose, mannitol and soluble starch were used as carbon sources in the basic enzyme production medium, glucose was the best carbon source for the enzyme production medium, and the laccase activity was 170.41±2.37U / ml. Glucose was used as the carbon source, and fermentation was carried out at an addition amount of 10, 20, 30, 40 and 50g / L to compare the effects of different concentrations of glucose on laccase production.

[0078] According to the above results, the seed liquid of XH005 strain was inoculated into the basic enzyme production medium at a rate of 3%, and glucose was added to the basic medium at different addition amounts (10.0, 20.0, 30.0, 40.0, 50 g / L) as the carbon source to investigate the effect of different addition amounts of glucose on the enzyme production of XH005. The culture was carried out at 30°C and 150 r / min for 8 days, and the enzyme activity of the fermentation supernatant was measured to determine the optimal addition amount of yeast extract powder.

[0079] Results Figure 6 .Depend on Figure 6 It can be seen that with the increase of glucose addition, the production of laccase first increased and then decreased. 40g / L of glucose was the optimal addition amount, and the laccase activity was 195.64±5.65U / ml.

[0080] 1.3.2 Effect of nitrogen source type and addition amount on laccase production

[0081] The seed liquid of strain XH005 was inoculated into the basic enzyme production medium at an inoculum size of 3%, and 4 g / L of organic nitrogen peptone, yeast extract powder, corn flour, ammonium chloride and ammonium sulfate were added as nitrogen sources. Other conditions remained unchanged and the culture was carried out at 30°C and 150 r / min for 8 days. The supernatant of the fermentation liquid was taken to determine the enzyme activity, the effects of different nitrogen sources on the enzyme production of XH005 were investigated, and the optimal nitrogen source was determined.

[0082] Results Figure 7 .Depend on Figure 7 It can be seen that when peptone, yeast extract powder, ammonium chloride, ammonium sulfate and corn flour were used as nitrogen sources in the basic enzyme production medium, yeast extract powder was the best nitrogen source for the enzyme production medium, and the laccase activity was 229.30±4.90U / ml. With yeast extract powder as the nitrogen source, fermentation was carried out at an addition amount of 2.0, 4.0, 6.0, 8.0 and 10.0g / L, and the effects of different concentrations of glucose on laccase production were compared.

[0083] According to the above results, the seed liquid of XH005 strain was inoculated into the basic enzyme production medium at an inoculation rate of 3%, and yeast extract powder was used as a nitrogen source, and added to the basic medium at different addition amounts (2.0, 4.0, 6.0, 8.0, 10.0 g / L) to investigate the effect of different addition amounts of yeast extract powder on the enzyme production of XH005. The culture was carried out at 30°C and 150 r / min for 8 days, and the enzyme activity of the fermentation liquid supernatant was measured to determine the optimal addition amount of yeast extract powder.

[0084] Results Figure 8 .Depend on Figure 8 It can be seen that with the increase in the amount of yeast extract powder added, the production of laccase first increased and then decreased. 6g / L yeast extract powder was the optimal addition amount, and the laccase activity was 262.41±2.36U / ml.

[0085] 1.3.3 Effect of inoculation amount on laccase production

[0086] The seed solution of strain XH005 was inoculated with different volume concentrations (1, 3, 5, 7, 9%) at 30°C and 150 r / min for 8 days, and the fermentation supernatant was taken to measure the enzyme activity to determine the optimal inoculation amount for enzyme production. Fig. 9 .Depend on Fig. 9 It can be seen that with the increase of inoculation amount, the enzyme activity increases rapidly. When the inoculation amount is 3%, the highest laccase activity of the fermentation broth is measured to be 169.23±3.79U / ml. However, when the strain inoculation amount is greater than 3%, the enzyme activity decreases with the increase of inoculation amount. It can be seen from the observation of the bacterial liquid that this may be because when the inoculation amount is too large, the viscosity of the culture broth increases, which can easily cause insufficient oxygen supply to the solution and rapid consumption of nutrients, resulting in malnutrition of mycelium, and a decrease in laccase activity. Therefore, the optimal inoculation amount for fermentation to produce laccase is 3%.

[0087] 1.3.4 Effects of metal ion type and addition amount on laccase production

[0088] The XH005 strain seed solution was inoculated into the basic enzyme production medium at a rate of 3%, and 1 mmol / L Cu 2+ , Fe 2+ , Ca 2+ , Mn 2+ and Zn 2+ Keeping other conditions unchanged, culture at 30℃ and 150r / min for 8 days, take the supernatant of the fermentation liquid to measure the enzyme activity and determine the best metal ion for enzyme production.

[0089] Results Fig.10 .Depend on Fig.10It can be seen that when anhydrous calcium chloride, CuSO4·5H2O, MnSO4·H2O, ZnSO4·7H2O, and FeSO4·7H2O were used as metal ions in the basic enzyme production medium, CuSO4·5H2O was the best ion for the enzyme production medium, and the laccase activity was 242.05±3.54U / ml. Yeast extract powder was used as the nitrogen source, and fermentation was carried out at 0.5, 1.0, 1.5, 2.0, and 2.5mmol / L additions to compare the effects of different concentrations of copper ions on laccase production.

[0090] According to the above results, the seed solution of strain XH005 was inoculated into the basic enzyme production medium at a rate of 3%, and Cu 2+ Different addition amounts (0.5, 1, 1.5, 2.0, 2.5 mmol / L) were added to the basal medium to investigate the effect of different addition amounts of glucose on the enzyme production of XH005. The culture was carried out at 30°C and 150 r / min for 8 days, and the enzyme activity was measured in the fermentation supernatant to determine the Cu 2+ Optimal addition amount.

[0091] Results Fig.11 .Depend on Fig.11 It can be seen that with the increase of the amount of metal ions added, the production of laccase first increased and then decreased. 1mmol / L of copper ions was the optimal addition amount, and the laccase activity was 240.26±5.77U / ml.

[0092] 1.3.5 Effect of pH on laccase production

[0093] The seed liquid of XH005 strain was inoculated with 3% in the basic enzyme production medium, and other conditions were kept unchanged. Different initial pH values ​​were set to 4.0, 5.0, 6.0, 7.0, and 8.0, and cultured at 30°C and 150r / min for 8 days. The enzyme activity was measured by taking the supernatant of the fermentation liquid. The effect of different initial pH values ​​on the enzyme activity of the strain was investigated, and the optimal enzyme production pH was determined to be 5.

[0094] Results Fig.12 .Depend on Fig.12 It can be seen that with the increase of pH, the production of laccase first increased and then tended to be stable. Although there were fluctuations, the fluctuations were small. pH = 5 was the best, and the laccase activity was 168.23 ± 9.44 U / ml.

[0095] 1.3.6 Box-Behnken test optimization

[0096] According to the results of the single factor experiment, four factors were selected, namely, glucose addition (A), yeast extract powder addition (B), inoculation amount (C), and copper ion addition (D). The laccase activity was used as the response value, and -1, 0, and 1 represented low, medium, and high levels, respectively. The Box-Behnken response surface optimization experiment was designed. The experimental design and experimental results are shown in Table 1. The design was carried out using Design-Expert 13 software.

[0097] Table 1 Response surface experiment results

[0098]

[0099]

[0100] A total of 29 experimental combinations were designed using Design Expert software, and the experimental results were fitted with a quadratic polynomial regression to obtain the regression equation of laccase activity Y for independent variables A, B, C, and D: Y = 468.96-12.68A+4.35B+9.48C-49.76D-29.56AB-5.68AC-3.51AD+29.90BC+39.76BD-11.04CD-106.24A 2 -81.78B 2 -55.18C 2 -83.55D 2 According to the results of the response surface test, the model was subjected to variance analysis and significance analysis, and the results are shown in Table 2.

[0101] Table 2 Response surface experiment results

[0102]

[0103]

[0104] The model p<0.0001, indicating that there is a very significant linear relationship between the independent variable and the dependent variable. The P value of the lack of fit term is 0.0634>0.05, which is not significant, proving that the model is effective. In addition, the R 2 The value is 0.9670, and the correction coefficient R 2 The Adj value was 0.9340, indicating that the model fitting degree of the regression equation was high and the model could well predict the relationship between the fermentation conditions of the strain and the enzyme activity.

[0105] The coefficients of the quadratic terms in the equation are all negative, the parabola of the model opens downward, and the equation has a maximum point. The absolute value of the coefficient of the linear term in the regression equation determines the order of the four factors affecting the laccase activity. In this study, the order of the four factors affecting the enzyme activity of XH005 is: glucose concentration (A)> copper ion concentration (D)> yeast extract powder concentration (B)> inoculation amount (C).

[0106] 2.3 Response surface analysis

[0107] The shape of the contour line can intuitively show the strength of the interaction between the two factors. The more the shape of the contour line is elliptical, the stronger the interaction between the two factors is, and the closer the shape is to a circle, the weaker the interaction between the two factors is. The pairwise interaction analysis of the four influencing factors of glucose addition (A), yeast extract powder addition (B), inoculation amount (C), and copper ion concentration (D) was performed, and the pairwise interaction contour line map was drawn. Fig.14 , Fig.16 and Fig.18 As shown, the contour lines of glucose addition amount and copper ion concentration, inoculum size and glucose addition amount, inoculum size and copper ion concentration are circular, proving that the interaction between the two is not strong.

[0108] The slope and steepness of the response surface can reflect the influence of various factors on the laccase activity. The steeper the curve, the more significant the influence of the factor on the enzyme activity of the strain; the flatter the curve, the smaller the influence on the enzyme production of the strain. Fig.13 It can be seen that when the amount of glucose added (A) remains unchanged, the enzyme activity of the strain increases first and then decreases with the increase of the amount of yeast extract powder added (B); when the amount of yeast extract powder added (B) remains unchanged, the enzyme activity of the strain changes consistently with the amount of glucose added (A). Fig.15 It can be seen that when the inoculation amount (C) remains unchanged and the amount of yeast extract powder added (B) increases, the enzyme activity of the fermentation broth shows a trend of first increasing and then decreasing; when the amount of yeast extract powder added (B) remains unchanged, the increase or decrease in the enzyme activity of the strain is consistent with the change range of the inoculation amount (C). Fig.17 It can be seen that when the amount of yeast extract powder added (B) remains unchanged, the enzyme activity increases first and then decreases with the increase of copper ion concentration (D); when the copper ion concentration (D) remains unchanged, the increase or decrease of the enzyme activity of the strain is consistent with the change in the amount of yeast extract powder added (B). The three-dimensional curves of the three groups of two factors AB, BC, and BD are steeper and the contour lines are elliptical, indicating that they have a significant effect on the enzyme activity of the P9 strain.

[0109] 2.3 Response surface optimization results and verification experiments

[0110] The optimal laccase production medium was analyzed by Design-Expert 13 software. When the addition of glucose was 39.45 (g / L), yeast extract powder was 5.97 (g / L), inoculation was 3.23%, and copper ion addition was 0.85 (mmol / L), the predicted maximum enzyme activity was 477.49U / mL. Three repeated verification experiments were carried out according to the conditions of the predicted results, and the enzyme activity of the strain was measured to be 480.22±7.53U / mL, which was close to the predicted value, and the relative standard deviation RSD was 1.57%, indicating that the parallel verification experiment had good repeatability. The enzyme activity of the optimized strain was 2.80 times higher than that of the previous enzyme production, proving that the model can better predict the relationship between fermentation conditions and enzyme activity of the strain, and is feasible for optimizing the fermentation enzyme production conditions of the bacteria.

[0111] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pleurotus ostreatus XH005, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.41421.

2. The culture of Pleurotus ostreatus XH005 according to claim 1.

3. A bacterial agent, characterized in that: Including the Pleurotus ostreatus XH005 described in claim 1.

4. Use of Pleurotus ostreatus XH005 according to claim 1, the culture according to claim 2 or the microbial agent according to claim 3 in producing laccase and / or degrading lignocellulose.

5. A method for producing laccase, characterized in that: The following steps are involved: The seed liquid of Pleurotus ostreatus XH005 according to claim 1 is inoculated into a culture medium and cultured to obtain a culture containing laccase.

6. The method according to claim 5, characterized in that The culture medium includes a first enzyme-producing culture medium and / or a second enzyme-producing culture medium; the first enzyme-producing culture medium uses water as a solvent and includes components with the following concentrations: 10 to 50 g / L of glucose, 2 to 10 g / L of yeast extract powder, and 0.5 to 2.5 mmol / L of copper sulfate; or the second enzyme-producing culture medium uses water as a solvent and includes components with the following concentrations: 30 g / L of glucose, 4 g / L of peptone, and 1.5 g / L of magnesium sulfate.

7. The method according to claim 6, characterized in that The concentration of glucose in the first enzyme production medium is 39.45-40 g / L.

8. The method according to claim 6, characterized in that The concentration of yeast extract in the first enzyme production medium is 5.97-6 g / L.

9. The method according to claim 6, characterized in that The concentration of copper sulfate in the first enzyme production culture medium is 0.85-1 mmol / L.

10. The method according to claim 5, characterized in that The effective spore number of Pleurotus ostreatus in the seed solution is 1×10 8 / mL; the inoculation amount of the seed liquid accounts for 1% to 5% of the total volume of the culture medium; and the culture time is 7 to 9 days.

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