Laccase-producing penicillium and application thereof

Abstract

The invention discloses laccase-producing penicillium and application thereof, and relates to the technical field of microbial fermentation. The invention provides a laccase-producing natural penicillium, the laccase-producing natural penicillium is identified and named as Penicilliumucsense, a culture medium of the laccase-producing natural penicillium is optimized, and culture conditions for efficiently producing laccase are obtained. Compared with an engineering strain for biologically synthesizing laccase, the strain has relatively strong environmental adaptability, laccase synthesis can be naturally performed under wide natural conditions, the genome of the strain is relatively stable, potential risks of exogenous genes do not exist, and the safety is relatively high. The penicillium is short in growth cycle, high in yield in the fermentation process and high in lignin degradation activity. The strain Penicilliumuse is applied to degradation of phenol-polluted soil, the toxicity reduction effect is remarkable, the degradation cost is reduced, the degradation period is shortened, and an excellent strain is provided for production of composite oxidase systems such as laccase.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a strain of Penicillium laccase-producing fungus and its applications. Background Technology

[0002] In the field of environmental pollutant degradation, the efficient decomposition of complex organic compounds such as lignin and polycyclic aromatic hydrocarbons has always been a major challenge. These substances possess highly stable aromatic ring structures, making them difficult to completely degrade using traditional physicochemical methods and prone to generating secondary pollution. In nature, microorganisms achieve efficient transformation of these stubborn substances through secreted oxidase systems, with laccase forming a key enzyme catalytic system. As a multi-copper oxidase, laccase catalyzes the oxidation of phenolic and non-phenolic substrates through a single-electron transfer mechanism, while simultaneously reducing molecular oxygen to water. This characteristic enables laccase to play a central role in the degradation of lignin and dyes.

[0003] Currently, microbial fermentation is the main method for the industrial production of laccase, and its core lies in screening high-laccase-producing strains or constructing engineered strains to enhance laccase synthesis capabilities. To optimize the metabolic pathways of strains and increase laccase yield, existing technologies often employ genetic engineering techniques to introduce exogenous plasmids into host strains, artificially modifying the metabolic pathways of the strains to construct engineered laccase biosynthetic strains. However, the aforementioned plasmid-based strain modification methods have significant technical drawbacks: Firstly, the introduction of exogenous plasmids disrupts the genetic integrity of the strain, leading to decreased genetic stability. During long-term subculturing or practical applications, plasmid loss and metabolic pathway disruptions are highly likely, thus affecting the continuity and stability of laccase production. Secondly, engineered strains, due to artificial genetic modification, have weak environmental adaptability and struggle to grow stably in complex natural environments, failing to meet the demands of practical applications such as pollutant degradation under natural conditions. Furthermore, the exogenous genes introduced into engineered strains pose a potential risk of diffusion, potentially disrupting biodiversity in natural ecosystems, and their biosafety needs further improvement. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a laccase-producing Penicillium strain that can naturally synthesize laccase and has a strong ability to adapt to the environment, without the potential risk of exogenous genes.

[0005] The technical problem to be solved by the present invention is to provide the application of the above-mentioned laccase-producing Penicillium in the preparation of laccase.

[0006] The technical problem to be solved by the present invention is to provide a method for producing laccase efficiently, which is obtained by fermentation of the above-mentioned laccase-producing Penicillium.

[0007] The technical problem to be solved by the present invention is to provide the application of the laccase prepared above in the degradation of lignin and / or dyes.

[0008] To address the aforementioned technical problems, this invention provides a laccase-producing *Penicillium laccasei* strain, which was screened from coking waste liquid and identified by 18S rRNA. Penicillium ucsense It was named Pu-lac.

[0009] Accordingly, the application of the aforementioned laccase-producing Penicillium in the preparation of laccase was also disclosed.

[0010] Accordingly, a method for producing laccase efficiently is also disclosed, which uses the aforementioned laccase-producing Penicillium to produce laccase through fermentation.

[0011] As an improvement to the above technical solution, a method for efficiently producing laccase includes the following steps: (1) The laccase-producing Penicillium laccase solution described in claim 1 is inoculated into MM medium for the first culture; (2) After adding sterile distilled water to the culture medium after culturing in step (1), scrape off the spores to prepare a spore suspension; (3) The diluted spore suspension was inoculated into the fermentation broth and cultured for a second time to obtain the fermentation broth; (4) Centrifuge the fermentation broth, discard the precipitate, and obtain laccase solution.

[0012] As an improvement to the above technical solution, in step (1), the temperature for the first culture is 35℃~37℃ and the time is 48h~96h.

[0013] As an improvement to the above technical solution, in step (1), the components of the MM culture medium, calculated by mass percentage, include 0.5%~1.5% glucose and 1 mol / L Mg. S O4 mother liquor 0.1%~0.3%, casein hydrolysate 0.1%~1.5%, yeast extract 0.2%~0.7% and ABTS 0.01%~0.05%.

[0014] As an improvement to the above technical solution, in step (3), the temperature for the second culture is 35℃~37℃ and the time is 72h~120h.

[0015] Accordingly, a laccase is also disclosed, which is prepared using the above-described efficient laccase production method.

[0016] Correspondingly, the application of laccase in the degradation of lignin was also disclosed.

[0017] Accordingly, the application of laccase in the degradation of dyes, including crystal violet and malachite green, is also disclosed.

[0018] Implementing this invention has the following beneficial effects: 1. This invention solves the problem of low yield and numerous byproducts in existing microbial fermentation processes for producing laccase, which increases the cost of subsequent laccase separation and purification. The Penicillium strain of this invention produces a series of oxidases during shake-flask fermentation that work in conjunction with laccase to degrade toxic products for application. Compared to genetically modified strains, the enzyme activity for degrading lignin is as high as 125.6 U / L.

[0019] 2. This invention solves the problem of genetic instability in existing microbial fermentation processes for producing laccase, which require the introduction of plasmids to modify the metabolic pathways of the strain. The Penicillium strain of this invention is a natural strain with strong environmental adaptability, capable of stable growth in natural environments. It can form biofilms on various surfaces, which not only enhances its environmental adaptability but also improves its resistance to antibiotics.

[0020] 3. Based on the natural characteristics of Penicillium's high oxidase production, this invention significantly improves the laccase production efficiency of the Penicillium strain through microbial fermentation optimization. The yield after 96 hours of shake-flask fermentation increased from 21.7 U / L to 125.6 U / L compared to the starting strain, providing an excellent strain for the preparation of laccase by microbial fermentation.

[0021] 4. The optimized natural laccase-producing Penicillium of this invention has an effect on the degradation of different dyes, providing a new direction for the degradation of toxins in fuel decolorization. Attached Figure Description

[0022] Figure 1 This is a gel image of the PCR electrophoresis products in Example 1 of the present invention; Figure 2 This is a graph showing the growth analysis of the Penicillium strain at different temperatures in Example 2 of the present invention; Figure 3 This is a graph showing the relative enzyme activity of laccase during fermentation in Example 2 of the present invention. Figure 4 This is a graph showing the optimal temperature and pH of laccase in Example 3 of the present invention. Figure 5 The degradation results of different dyes by laccase in Example 4 of this invention are shown. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0024] Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Raw materials whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0025] This embodiment discloses a laccase-producing Penicillium strain, which was screened from coking waste liquid and identified by 18S rRNA. Penicillium ucsense It was named Pu-lac.

[0026] It is worth noting that, compared with engineered strains used to construct biosynthetic laccase, the laccase-producing *Penicillium* strain of this embodiment has a stronger environmental adaptability, can naturally synthesize laccase under a wide range of natural conditions, and its genome is relatively stable, without the potential risk of exogenous genes, thus exhibiting relatively high safety. This *Penicillium* strain has a short growth cycle, high yield during fermentation, and high lignin-degrading activity. After 72 hours of fermentation, the lignin-degrading activity can reach 125.6 U / L. The strain of this invention... Penicillium ucsense When applied to the degradation of phenol-contaminated soil, it has a significant detoxification effect, shortens the degradation cost and cycle, and provides an excellent strain for the production of complex oxidase systems such as laccase.

[0027] Further explanation reveals that *Penicillium* possesses several advantages. It produces a large number of spores with high resilience, capable of surviving for extended periods under extreme environmental conditions (including high temperature, low temperature, dryness, acidity, alkalinity, high salinity, high pressure, ultraviolet radiation, and disinfectants). These spores can form biofilms on various surfaces, enhancing their environmental adaptability and antibiotic resistance. They have broad application potential in various fields: *Penicillium* is a saprophytic fungus that, in addition to laccase, secretes ligninase and cellulase, utilizing these extracellular enzymes to decompose organic matter into simple small molecules such as glucose and amino acids for its own use. Some *Penicillium* strains can degrade various organic pollutants and heavy metals, such as petroleum hydrocarbons and phenolic compounds, thus having important applications in environmental remediation and pollution control. *Penicillium* can also maintain its survival by altering its metabolic rate under different environmental conditions, thus improving its resilience. The laccase-producing *Penicillium* strain of this invention possesses the ability to produce abundant oxidase systems and exhibits strong adaptability, high safety, and good stability. The strain is inexpensive, readily available, and requires minimal culture conditions, making it an excellent strain for laccase production.

[0028] Specifically, coking waste liquid is a non-degradable organic waste liquid generated during the production of coke and coal tar. It contains strong carcinogens such as polycyclic aromatic hydrocarbons, has excessive nitrogen content, and high pollutant concentrations.

[0029] Preferably, the coking waste liquid is collected from Jinma Coking Plant in Hancheng City, Shaanxi Province. Specifically, Jinma Coking Plant in Hancheng City, Shaanxi Province is Hancheng Jinma Coking Co., Ltd.

[0030] Accordingly, this embodiment also discloses the application of the above-mentioned laccase-producing Penicillium in the preparation of laccase.

[0031] Accordingly, this embodiment also discloses a method for producing laccase efficiently, which is produced by fermentation using the laccase-producing Penicillium as described in claim 1.

[0032] In some embodiments, the method for efficiently producing laccase includes the following steps: (1) The laccase-producing Penicillium laccase solution described in claim 1 is inoculated into MM medium for the first culture; specifically, MM medium is a basic culture medium commonly used in microbiology, which contains the basic nutrients required for microbial growth; (2) After adding sterile distilled water to the culture medium after culturing in step (1), scrape off the spores to prepare a spore suspension; (3) Inoculate the diluted spore suspension into the fermentation culture medium and carry out a second culture to obtain the fermentation broth; (4) Centrifuge the fermentation broth, discard the precipitate, and obtain laccase solution.

[0033] In some implementations, in step (1), the temperature for the first culture is 35°C to 37°C and the time is 48h to 96h.

[0034] In some embodiments, in step (1), the MM culture medium comprises, by mass percentage, 0.5%–1.5% glucose and 1 mol / L Mg. S O4 mother liquor 0.1%~0.3%, casein hydrolysate 0.1%~1.5%, yeast extract 0.2%~0.7% and ABTS 0.01%~0.05%.

[0035] In one embodiment, the MM medium comprises 1% glucose and 1 mol / L Mg. S O4 mother liquor 0.2%, casein hydrolysate 0.1%, yeast extract 0.5% and ABTS 0.01%.

[0036] In some embodiments, in step (3), the temperature for the second culture is 35°C to 37°C and the time is 72h to 120h.

[0037] Accordingly, this embodiment also discloses a laccase prepared using the above-described efficient laccase production method.

[0038] Accordingly, this embodiment also discloses the application of the above-mentioned laccase in the degradation of lignin.

[0039] Accordingly, this embodiment also discloses the application of the above-mentioned laccase in the degradation of dyes, including crystal violet and malachite green.

[0040] The technical solution of the present invention is further illustrated below through embodiments.

[0041] Example 1 This embodiment provides a laccase-producing Penicillium strain, which was screened from coking waste liquid samples collected from Jinma Coking Plant in Hancheng City, Shaanxi Province. After optimization by microbial fermentation, a highly efficient laccase-producing Penicillium strain was obtained and named Pu-lac.

[0042] Specifically, the screening method for laccase-producing Penicillium includes the following steps: I. Isolation and Screening of Strains The strain Pu-lac in this embodiment was isolated from coking waste liquid samples collected from Jinma Coking Plant in Hancheng City, Shaanxi Province, and was obtained by three separate purifications on PDA medium using the three-zone streak method.

[0043] The specific screening method for strains includes the following steps: S1. Take 2g of coking waste liquid sample from Jinma Coking Plant in Hancheng, Shaanxi Province and add it to 20mL of sterile enrichment medium. Incubate at 35℃~37℃ and 180rpm~200rpm for 24h. S2. Take 1 mL of bacterial suspension for enrichment culture, and then perform 10 [units of culture] on the enriched bacterial solution. -1 ~10 -5 Serial dilutions were performed, and 200 μL of each dilution was spread onto PDA medium plates containing 0.01 ABTS and incubated at 35–37 °C for 72 h. S3. Select single colonies with good growth morphology, moderate size and obvious dark green hydrolysis zone, and isolate them by streaking multiple times. Inoculate them into sterile MM liquid medium and incubate at 35℃~37℃ and 180rpm~200rpm for 72h. S4. Take fresh bacterial culture that has been cultured for 24 hours, perform PCR amplification using 18S primers, and determine the 18S rRNA sequence after electrophoresis detection of the PCR product.

[0044] II. Identification of the strain 1. Identification of physiological and biochemical characteristics Pu-lac strain in MM (1% glucose, 1 mol / L Mg) SOn a solid culture medium containing 0.2% O4 stock solution, 0.1% casein hydrolysate, 0.5% yeast extract, and 0.01% ABTS, colonies are round or irregular in shape, with dense, rope-like hyphae on the surface. The colonies are smooth and moist, with neat edges, a viscous texture, and a pea-green color, exhibiting a certain luster and elasticity. Available carbon sources include fructose, maltose, sucrose, starch, arabinose, and cellulose; available nitrogen sources include ammonium sulfate, ammonium chloride, potassium nitrate, urea, and yeast extract.

[0045] 2. 18S rRNA sequence identification The activated strain Pu-lac was inoculated into liquid MM medium and cultured at 37°C with shaking at 180 rpm for 24 h. 1 μL of the bacterial culture was used as a template to amplify the 18S rDNA gene using universal primers 18S-F: 5'-CCAGTAGTCATATGCTTGTCT-3' and 18S-R: 5'-ACCTTGTTACGACTTTTACTTCC-3'. 2 μL of the PCR product was verified by 1% agarose gel electrophoresis. The specific PCR product electrophoresis gel image is shown below. Figure 1 As shown.

[0046] Based on the identification results and sequence alignment, strain Pu-lac was determined to belong to the genus *Penicillium*. Comparison of strain Pu-lac with NCBI data confirmed its identity as *Penicillium*, with a search coverage of 99%.

[0047] Example 2 A method for efficiently producing laccase, characterized by comprising the following steps: (1) The Pu-lac bacterial culture stored at -80℃ was streaked onto MM medium and cultured in an incubator at 35℃~37℃ for 72h; (2) After adding sterile distilled water to the culture medium after culturing in step (1), scrape off the spores to prepare a spore suspension and dilute it; (3) The diluted spore suspension was inoculated into the fermentation broth and cultured in an incubator at 35℃~37℃ for 120h to obtain the fermentation broth; the relative enzyme activity of laccase was analyzed during the fermentation process, and the analysis of the relative enzyme activity of laccase during the fermentation process is as follows: Figure 3 As shown; (4) Centrifuge the fermentation broth, discard the precipitate, and obtain laccase solution.

[0048] Specifically, a portion of the diluted spore suspension was spread onto MM plates and placed in incubators at 25℃, 28℃, 30℃, 37℃, and 42℃, respectively. The growth status was observed and recorded every 24 hours. Figure 2 As shown.

[0049] Example 3 The properties of the laccase prepared in Example 2 were determined. Determination of optimal pH: Prepare substrate buffer solutions with a volume fraction of 1% at pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, and 9.0. Mix the enzyme solution and substrate at a ratio of 1:19 (V:V) and incubate the mixture at the optimal temperature for 10 min. Measure the absorbance at 420 nm and calculate the relative enzyme activity. Results are as follows: Figure 4 As shown in -A, the enzyme activity remains above 80% within the pH range of 3.0 to 4.5.

[0050] Determination of optimal temperature: The enzyme solution and substrate were mixed in a 1:19 (V:V) reaction system and reacted at 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃ for 10 min, respectively. The absorbance at 420 nm was measured, and the relative enzyme activity was calculated. The results are as follows: Figure 4 As shown in -B, the optimal reaction temperature is 35℃, and at 50℃, the enzyme activity decreases to 62%.

[0051] After confirming the optimal pH, a pH stability experiment was conducted: the enzyme solution was stored at the optimal temperature and at pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 for 1 hour, with the untreated solution serving as the control group. The relative enzyme activity was calculated. The results are as follows: Figure 4 As shown in -C, the enzyme reaches its peak relative activity (approximately 100%) at pH 3.0, and retains more than 70% of its activity in the pH range of 2.0 to 7.0, indicating that it has good pH stability in the pH range of 2.0 to 7.0.

[0052] After confirming the optimal temperature, a temperature stability experiment was conducted: the enzyme solution was incubated at the optimal pH of 35℃ for 1 hour, and then reacted with the substrate under optimal conditions for 10 minutes. The OD value was measured at 420 nm. The results are as follows: Figure 4 As shown in Figure -D, after treatment at 35℃ for 60 min, the enzyme activity was retained by about 80%; while after treatment at 40℃ for 60 min, the enzyme activity was retained by only about 50%, indicating that the enzyme has good temperature stability at 35℃.

[0053] Example 4 This embodiment provides the application of laccase solution in the degradation of dyes, namely crystal violet and malachite green, and the application method includes: Enzyme solutions under optimal fermentation conditions were added to crystal violet and malachite green at different gradients, with the concentration of both crystal violet and malachite green being 20 mg·L⁻¹. The mixtures were then placed in a 30℃ constant temperature incubator for 48 h, and the absorbance of the dyes was measured.

[0054] The method for degrading crystal violet and malachite green with laccase solution includes the following steps: a. Using a visible light spectrophotometer to scan the spectrum, it can be determined that the maximum absorption wavelength of crystal violet is 591 nm; b. Prepare a crystal violet solution system with a concentration of 20 mg·L⁻¹, add 200 μL, 400 μL, 600 μL and 800 μL of enzyme solution respectively, and incubate in a constant temperature incubator at 28-30℃ for 48 h, and measure the absorbance of the dye. c. Using a visible light spectrophotometer to scan the spectrum, it can be found that the maximum absorption wavelength of malachite green is 616 nm; d. Prepare a malachite green solution system with a concentration of 20 mg·L⁻¹, add 200 μL, 400 μL, 600 μL, and 800 μL of crude enzyme solution respectively, and incubate in a constant temperature incubator at 28-30℃ for 48 h and 96 h respectively, and measure the absorbance of the dye.

[0055] The degradation results of crystal violet and malachite green in this embodiment are as follows: Figure 5 As shown.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A strain of laccase-producing Penicillium, characterized in that, The laccase-producing Penicillium was screened from coking waste liquid and identified by 18S rRNA. Penicilliumucsense It was named Pu-lac.

2. The application of the laccase-producing Penicillium as described in claim 1 in the preparation of laccase.

3. A method for efficiently producing laccase, characterized in that, It is produced by fermentation using the laccase-producing Penicillium as described in claim 1.

4. The method for efficiently producing laccase according to claim 3, characterized in that, Includes the following steps: (1) The laccase-producing Penicillium laccase solution described in claim 1 is inoculated into MM medium for the first culture; (2) After adding sterile distilled water to the culture medium after culturing in step (1), scrape off the spores to prepare a spore suspension; (3) The diluted spore suspension was inoculated into the fermentation broth and cultured for a second time to obtain the fermentation broth; (4) Centrifuge the fermentation broth, discard the precipitate, and obtain laccase solution.

5. The method for efficiently producing laccase according to claim 4, characterized in that, In step (1), the temperature for the first culture is 35℃~37℃ and the time is 48h~96h.

6. The method for efficiently producing laccase according to claim 4, characterized in that, In step (1), the components of MM medium, calculated by mass percentage, include 0.5%–1.5% glucose and 1 mol / L Mg. S O4 mother liquor 0.1%~0.3%, casein hydrolysate 0.1%~1.5%, yeast extract 0.2%~0.7% and ABTS 0.01%~0.05%.

7. The method for efficiently producing laccase according to claim 4, characterized in that, In step (3), the temperature for the second culture is 35℃~37℃ and the time is 72h~120h.

8. A laccase, characterized in that, It is prepared using the efficient laccase production method described in any one of claims 3-7.

9. The application of the laccase according to claim 8 in the degradation of lignin.

10. The use of the laccase of claim 8 in the degradation of dyes, wherein the dyes include crystal violet and malachite green.

Images