Fungus laccase OrLac5 from oudemansiella radicata as well as coding gene and application thereof

Abstract

The invention discloses fungus laccase OrLac5 from oudemansiella radicata as well as a coding gene and application of the fungus laccase OrLac5. The amino acid sequence of the fungus laccase OrLac5 is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene of the fungus laccase OrLac5 is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the fungus laccase OrLac5. The fungal laccase OrLac5 is resistant to acid, the optimum pH value is 3.0, and the fungal laccase OrLac5 has good stability in the pH value range of 2.0-4.0; the optimum temperature is 50 DEG C, the enzyme can tolerate high temperature, and 70% of enzyme activity can still be kept after the enzyme is treated at 60 DEG C for 12 hours; cu < 2 + >, Fe < 2 + >, Mg < 2 + > and Zn < 2 + > have a relatively strong promotion effect on fungal laccase OrLac5. The fungal laccase OrLac5 can be used for degrading anthraquinone dyes under the conditions of acidity, high temperature and the like, and particularly, the degradation rates of acid blue 40 and acid green 25 in 24 hours are 87% and 89% respectively. The fungal laccase OrLac5 can significantly improve the saccharification efficiency of wood chips, corncobs and cottonseed hulls. The laccase can be used in the fields of dye degradation treatment, improvement of the utilization efficiency of an edible mushroom matrix and the like, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a fungal laccase OrLac5 derived from Oyster mushroom and a coding gene and application thereof. Background Art

[0002] Agricultural and forestry waste is the main raw material for edible fungus cultivation, among which lignocellulose is the main biomass component. Lignocellulose uses cellulose as the skeleton, hemicellulose and pectin are combined between the cellulose, and lignin is wrapped in the outermost layer. It is the main carbon source for the growth of edible fungi. During the growth of edible fungi, extracellular enzymes are secreted to first depolymerize lignin, so that the internal cellulose and hemicellulose are exposed for further utilization. The degradation and utilization efficiency of lignocellulose affects the growth rate of mycelium. The higher the activity of the enzyme that decomposes lignocellulose, the more complete the mycelium decomposes the culture matrix, and the shorter the mushroom production time. According to literature reports, the activity of laccase in the mycelium growth stage significantly affects the growth of mycelium, thereby affecting the yield of edible fungi.

[0003] Oudesporium Oudemansiella raphanipes , commonly known as long root mushroom, trade name black skin Alpinia radiata) is a rare edible mushroom with great economic value, and has been loved by consumers in recent years. The cultivation substrate of long root mushroom is mainly sawdust, corn cobs and cottonseed hulls, but the growth cycle of long root mushroom is long, and improving the degradation efficiency of lignocellulose can shorten the growth cycle. The applicant found a laccase with high expression in the early growth and development of long root mushroom through transcriptome and proteomics technology, and speculated that it plays a key role in the decomposition of lignocellulose in long root mushroom. The protein was heterologously expressed and purified by molecular biology technology, and the role of this enzyme in lignocellulose degradation was verified.

[0004] Laccase is a type of copper-containing polyphenol oxidase that belongs to the lignin decomposing enzyme system. Laccase has a wide substrate spectrum and can catalyze aromatic compounds; it can catalyze the degradation of antibiotics and dyes; it can catalyze the conversion of lignocellulose. The fungal genome contains abundant laccase genes. There have been a lot of research reports on the treatment of textile wastewater by laccases mainly from white rot fungi. Laccase isolated from Ganoderma lucidum can effectively degrade two azo dyes, Remazol Brilliant Violet 5R (RBV5) and SunsetYellow (SY). Polyporus The laccase from sp. S133 had a good degradation effect on the anthraquinone dye RemazolBrilliant Blue R (RBBR). Perenniporia subacida and Coprinus comatusThe strains also have the ability to degrade anthraquinone dyes. The secretory proteins of Coprinus comatus have the ability to degrade trityl dyes, such as crystal violet and malachite green. However, these laccases need to add mediators (ABTS, HBT, etc.) to degrade dyes, which increases the processing cost and introduces new compounds. Summary of the invention

[0005] The purpose of the present invention is to provide a fungal laccase OrLac5 derived from Oyster mushroom and its encoding gene and application. The present invention separates a new fungal laccase OrLac5 from the secretory protein of Oyster mushroom, obtains the fungal laccase through gene cloning, heterologous expression, protein purification and other processes, and applies it to dye decolorization and improving the saccharification efficiency of lignocellulose.

[0006] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The invention provides a fungal laccase OrLac5 derived from Oyster mushroom, and the amino acid sequence of the fungus OrLac5 is shown in SEQ ID NO.1.

[0007] Furthermore, the fungal laccase OrLac5 has good enzyme activity stability in the pH range of 2.0-5.0, and the optimum pH is 3.0.

[0008] Furthermore, the optimal enzyme activity temperature of the fungal laccase OrLac5 is 50°C.

[0009] The present invention also provides a fungal laccase OrLac5 encoding gene fungal laccase gene OrLac5 , whose nucleotide sequence is shown in SEQ ID NO.2.

[0010] The present invention also provides a fungal laccase gene containing the OrLac5 recombinant vectors and recombinant strains.

[0011] Furthermore, the recombinant strain is Pichia pastoris.

[0012] The present invention also provides the fungal laccase OrLac5 or the fungal laccase gene OrLac5 Or the use of the recombinant vector and recombinant strain in dye decolorization.

[0013] Furthermore, the dyes include trityl dyes, anthraquinone dyes and azo dyes.

[0014] Furthermore, the anthraquinone dyes include acid blue 40 and acid green 25.

[0015] The present invention also provides the fungal laccase OrLac5 or the fungal laccase gene OrLac5Or the use of the recombinant vector and recombinant strain in improving the saccharification efficiency of lignocellulose.

[0016] Furthermore, the lignocellulose includes sawdust, corn cobs and cottonseed hulls.

[0017] Furthermore, the optimum pH for the application of the fungal laccase OrLac5 is 2.5, and the optimum reaction temperature is 50°C.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: the fungal laccase OrLac5 provided by the present invention is derived from Oyster mushroom and belongs to the extracellular enzyme of Oyster mushroom. Compared with the existing laccase, the fungal laccase has a certain degradation effect on three different types of dyes (trityl, anthraquinone and azo) without adding any medium, among which the degradation effect on anthraquinone dyes is better, especially the degradation efficiency of acid blue 40 and acid green 25 can reach 87%-89%. It can be used to remove anthraquinone dyes in the environment.

[0019] The fungal laccase OrLac5 is suitable for acidic conditions (optimum pH is 2.5), has good enzyme activity stability in the range of pH = 2.0 ~ 5.0; the optimal enzyme activity temperature is 50 ° C, can tolerate higher temperatures, and can still maintain more than 70% enzyme activity after being treated at 60 ° C for 12 hours. The fungal laccase of the present invention can degrade lignocellulose and improve the saccharification efficiency of sawdust, corn cobs and cottonseed hulls.

[0020] Therefore, the fungal laccase OrLac5 of the present invention can be used to remove dyes under acidic and high temperature conditions (without the need to add additional media), can be used in the saccharification process of lignocellulose, improve the utilization efficiency of edible fungi substrates, and shorten the growth cycle of edible fungi. It has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The SDS-PAGE electrophoretogram of the purified fungal laccase OrLac5; Figure 2 It shows that the fungal laccase OrLac5 is pH stable; Figure 3 It shows the effect of temperature on the activity of fungal laccase OrLac5; Figure 4 It showed that the fungal laccase OrLac5 was temperature stable (60°C); Figure 5 It shows the effect of metal ions on the activity of fungal laccase OrLac5; Figure 6 It shows that the fungal laccase OrLac5 has a degradation effect on dyes; Figure 7It shows the saccharification efficiency of fungal laccase OrLac5 on lignin; Figure 8 This indicates the saccharification efficiency of fungal laccase OrLac5 on cellulose. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0023] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies. In the following examples, all molecular biology operations are performed with reference to "Molecular Cloning Experiment Guide" (Science Press, 2002 edition).

[0024] MD medium: 13.4 g / L amino-free yeast nitrogen base (YNB), 0.4 mg / L biotin, 20 g / L glucose.

[0025] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L agar.

[0026] BMGY medium: 13.4 g / L amino-free yeast nitrogen base (YNB), 10 g / L yeast powder, 20 g / L peptone, 10 g / L glycerol, 0.01 mol / L (pH 6.0) phosphate buffer, and 0.4 mg / L biotin.

[0027] BMMY medium: 13.4 g / L amino-free yeast nitrogen base (YNB), 10 g / L yeast powder, 20 g / L peptone, 10 g / L glycerol, 0.01 mol / L (pH 6.0) phosphate buffer, 0.4 mg / L biotin, and 1% methanol.

[0028] Example 1: Cloning of fungal laccase gene and its expression and preparation in Pichia pastoris (1) Cloning of fungal laccase genes RNA from the mycelium of Oyster mushroom (commercially available) was extracted using the modified phenol method, and cDNA was obtained by reverse transcription using the HiScript Ⅲ 1st StrandcDNA Synthesis Kit. The designed cloning primers are shown in Table 1, and the obtained cDNA was used as a template for PCR amplification. The PCR reaction parameters were: 98°C 5 min; 98°C 30 s; 56°C 30 s, 72°C 60 s, 34 cycles; 72°C 5 min. A fragment of about 1100 bp was amplified, which was recovered and sent for sequencing to obtain the fungal laccase gene. OrLac5The sequencing results showed that the nucleotide sequence was as shown in SEQ ID NO.2.

[0029] Table 1 Primers required for cloning

[0030] (2) Construction of expression recombinant expression vector The obtained amplified fragment was connected to the pPIC9k exogenous expression vector linearized by EcoR Ⅰ and Not Ⅰ (TakaRa) by homologous recombination technology, and heat-shocked into Escherichia coli Trans-T1 cells to screen positive transformants. The correct recombinant plasmid pPIC9k-19793-Lac5 was obtained. The pPIC9k-19793-Lac5 recombinant plasmid was linearized with restriction endonuclease SacⅠ (Vazyme), and electroporated into Pichia pastoris GS115 competent cells (Yisheng Biotechnology (Shanghai) Co., Ltd.), inoculated into MD medium and cultured for 2-3 days. The cells were cultured at 30℃ for 2-3 days on YPD medium plates containing 0, 2, and 4 mg / mL geneticin (G418), respectively, and positive transformants were screened and sequenced using universal primers of pPIC9k (Shanghai Sangon Biotechnology Co., Ltd.).

[0031] (3) Inducible expression in yeast cells The correct transformants were transferred into BMGY medium and cultured at 30 °C with shaking at 200 rpm until OD600 = 2-6; The cells were centrifuged at 5,000 rpm for 5 minutes, and the cells were collected and washed three times with PBS buffer. The cells were transferred to 50 mL BMMY medium, and the initial OD600 was controlled to be 1.0. 1% methanol was added and cultured at 30 °C and 200 rpm with shaking. A final concentration of 1% methanol was added every 24 h to continuously induce the cells to produce enzymes. 1 mL of bacterial solution was taken, the OD600 was measured, and the cells were centrifuged at 12,000 rpm for 5 min. The supernatant was taken to determine the laccase activity, and the growth status of the cells and the enzyme production were analyzed. The induction was continued for 120 h.

[0032] (4) Purification of recombinant laccase After 5 days of induction of BMMY medium in a shake flask with 1% methanol as an inducer, a fermentation broth with laccase activity was obtained. The column was pre-equilibrated with PB buffer (50 mM pH=8.0, containing 0.5 M NaCl) containing 5 mM imidazole. After loading, 10 column volumes of PB buffer containing 10 mM imidazole were used to wash away impurities, and then the gel medium was washed with 20 mM imidazole buffer to elute the target protein. Desalting and concentration were performed by ultrafiltration to detect laccase activity. The protein purification effect was detected by SDS-PAGE, and the results were ( Figure 1) showed that the purified protein band was single and the size was consistent with the predicted value, which was the fungal laccase OrLac5.

[0033] Determination of laccase activity: Mix 100 mM malonic acid buffer (pH 4.5) and 1 mM ABTS solution (prepared with malonic acid-sodium malonate buffer pH 4.5), shake well, and place in a 30 °C water bath. Take 1 mL of the above reagent in a 1.4 mL cuvette and adjust to zero at 420 nm. Add 50 μL of the enzyme solution to be tested, mix well, and immediately record the absorbance value. Record once every 30 s for a total of 5 times, take the average value, and convert it into the absorbance change per minute. The molar absorption coefficient of the product formed by ABTS catalyzed by laccase is 36000 mol -1 ·L·cm -1 .

[0034] The amino acid sequence of the fungal laccase is shown in SEQ ID NO.1, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0035] Example 2: Enzyme activity characteristics of fungal laccase pH and temperature are important factors affecting the catalytic activity of enzymes. The catalytic activity of the fungal laccase in Example 1 of the present invention was detected using 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate. The maximum pH range for the catalytic activity detection of the fungal laccase of the present invention is 1.0~6.0; the temperature range for the catalytic activity detection of the fungal laccase of the present invention is 20~90°C. The enzyme activity detection method is shown in Example 1.

[0036] Optimum pH Using ABTS as substrate, the activity of fungal laccase was detected in buffers with different pH values ​​(1.0-6.0). Figure 2 As shown, fungal laccase is relatively stable in the pH range of 1.0-6.0, with the optimum pH being 2.5. When the pH value is lower or higher than this range, the activity decreases significantly. This indicates that fungal laccase has good pH stability under acidic conditions.

[0037] Optimum temperature Using ABTS as substrate, the activity of fungal laccase was detected under different temperatures (20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃). Figure 3 As shown, the optimum reaction temperature of laccase is 50℃, and it can tolerate higher temperatures and still have more than 50% enzyme activity at 70℃.

[0038] (3) Thermal stability Using ABTS as substrate, the enzymatic activity of fungal laccase was detected under the conditions of incubation at high temperature of 60℃ for different time (0, 2, 4, 6, 8, 10, 12, 14 h). Figure 4 As shown in the figure, after being treated at 60℃ for 12 h, laccase can still retain more than 70% of its relative enzyme activity, showing good temperature stability.

[0039] (4) Effects of metal ions on enzyme activity Using ABTS as substrate, the fungal laccase was detected in the presence of different metal ions (Ca 2+ 、Cd 2+ , Cu 2+ 、Co 2+ , Fe 2+ , Li 2+ Mg 2 + , Mn 2+ 、Mo 2+ 、Na + , K + 、Zn 2+ ) under the conditions of reaction laccase activity. The results are as follows Figure 5 As shown, Cu 2+ , Fe 2+ Mg 2+ 、Zn 2+ It has a strong promoting effect on laccase activity.

[0040] Example 3: Application of fungal laccase OrLac5 in dye degradation Nine dyes, namely Coomassie Brilliant Blue G250, Reactive Turquoise Blue, Crystal Violet, Reactive Blue KNR, Reactive Yellow, Acid Green 25, Methyl Orange, Bromophenol Blue and Acid Blue 40, were used as substrates. The detailed information of the dyes is shown in Table 2. The concentration of the dyes was 50 mg / L. 50 μL of purified fungal laccase (0.1 U) was added to 50 mM malonic acid buffer (pH 2.5) and placed in a shaker at 125 rpm and 25°C for 24 h. The blank control group was replaced with 50 μL of distilled water instead of enzyme solution. The absorbance values ​​of the blank control group and the reaction group were measured at the corresponding absorbance values, and the degradation rate was calculated based on the absorbance values ​​obtained. The calculation method is as follows: Degradation rate (%) = 1-(A2-A0) / (A1-A0) Where A0 is the absorbance value of distilled water under the absorbance values ​​corresponding to different dyes; A1 is the absorbance value of the control group of each dye; A2 is the absorbance value of the experimental group of each dye.

[0041] The calculation shows that the degradation efficiency of 0.1 U laccase on the initial 50 mg / L dye at 25℃ for 24 h is as follows: Figure 6As shown in the figure, without adding a medium, the decolorization rate of anthraquinone dyes by laccase was relatively high, especially for acid blue 40 and acid green 25, with the degradation rates of 87%-89% in 24 h, respectively.

[0042] Table 2 Dye types and detection wavelengths

[0043] Example 4: Application of fungal laccase OrLac5 in improving saccharification efficiency of lignocellulose The cultivation substrates of industrial-scale long root mushrooms are mainly sawdust, corn cobs and cottonseed hulls. Through transcriptome and proteome data analysis, the laccase is the protein with the highest expression in the early stage of mycelial growth and development. The enzyme activity of the laccase in the early stage of long root mushroom growth and development was further detected, and the results of the enzyme activity detection were consistent with the results of the transcriptome and proteome. It is speculated that the laccase is mainly involved in the saccharification process of lignin cellulose. Therefore, the amount of reducing sugar after the fungal laccase OrLac5 was treated with sawdust, corn cobs and cottonseed hulls was detected, and the saccharification efficiency was calculated.

[0044] Pretreatment of sawdust, corn cobs and cottonseed hulls: The raw materials are dried and crushed, filtered through a 50-80 mesh sieve and set aside.

[0045] 40 mg of sawdust / corn cob / cottonseed hull was directly treated with 30 U of enzyme solution at 50 °C. After 48 h, the reducing sugar contents were 7.21 mg, 4.87 mg, and 5.93 mg, respectively, as determined by DNS method. Figure 7 ) were 17.45%, 12.17% and 14.825% respectively. The content of reducing sugar was determined by DNS method. For specific methods, please refer to https: / / protocols.mushroomlab.cn / archives / reducingsugar-dns.

[0046] Then, sawdust, corncobs, and cottonseed hulls were treated with alkali (5% NaOH for 1 hour) and high pressure (121°C for 30 minutes) to remove lignin from the materials, rinsed with distilled water until the pH no longer changed, and dried for later use. 40 mg of sawdust / corncobs / cottonseed hulls that had been delignined were treated with 30 U of enzyme solution at 50°C. After 48 hours, the reducing sugar contents detected by the DNS method were 7.45 mg, 12.03 mg, and 9.87 mg, respectively. Saccharification efficiency ( Figure 8 ) were 18.625%, 28.026% and 24.675% respectively. The content of reducing sugar was determined by DNS method. For specific methods, please refer to https: / / protocols.mushroomlab.cn / archives / reducingsugar-dns.

[0047] The present invention illustrates through the above experimental results that the fungal laccase OrLac5 provided by the present invention can effectively degrade lignin, cellulose and hemicellulose in sawdust, corn cobs and cottonseed hulls, etc., improve the substrate utilization efficiency of edible fungi such as Oyster mushroom, shorten the growth cycle of the strain, and has good application prospects.

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A fungal laccase OrLac5 derived from Oyster mushroom, characterized in that: The amino acid sequence of the fungal laccase OrLac5 is shown in SEQ ID NO.

1.

2. The coding gene of the fungal laccase OrLac5 according to claim 1 Fungal laccase gene OrLac5 , characterized in that: The fungal laccase gene OrLac5 The nucleotide sequence is shown in SEQ ID NO.

2.

3. The fungal laccase gene according to claim 2 OrLac5 , characterized in that: The fungal laccase gene OrLac5 The amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. Containing the fungal laccase gene according to claim 2 OrLac5 recombinant vectors and recombinant strains.

5. The fungal laccase OrLac5 of claim 1 or the fungal laccase gene of claim 2 OrLac5 Or use of the recombinant vector or recombinant strain according to claim 4 in dye decolorization.

6. The use according to claim 5, characterized in that: The dyes include trityl dyes, anthraquinone dyes and azo dyes.

7. The use according to claim 6, characterized in that: The anthraquinone dyes include acid blue 40 and acid green 25.

8. The fungal laccase OrLac5 of claim 1 or the fungal laccase gene of claim 2 OrLac5 Or the use of the recombinant vector and recombinant strain according to claim 4 in improving the saccharification efficiency of edible fungus matrix.

9. The use according to claim 8, characterized in that: The edible fungus substrate comprises wood chips, corn cobs and cottonseed hulls.

10. The use according to claim 5 or 8, characterized in that: The optimum pH for the application of the fungal laccase OrLac5 is 3.0, and the optimum reaction temperature is 50°C.

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