Laccase and application thereof

Abstract

The invention discloses laccase and application thereof, and belongs to the field of bioengineering. The high-activity laccase Lac-11 is obtained through database mining, and compared with existing high-activity laccase BstCotA, the high-activity laccase Lac-11 has the following beneficial effects that 1, the enzyme activity is remarkably improved, the enzyme activity is improved by nearly 50% under the conditions that the temperature is 37 DEG C and the pH is 5.0, and the industrial enzyme cost is reduced; 2, the pH stability is higher, the enzyme activity retention rate is higher in the pH range of 5.0-8.0, and the method is suitable for wider acid-base environments. And 3, the thermal stability is better, the stability at the medium-high temperature interval of 50-80 DEG C is obviously higher than that of a control enzyme, and the method is suitable for an industrial high-temperature process. 4, the dye decolorization efficiency is higher, the decolorization rates of amino black 10B and crystal violet respectively reach 31% and 37%, and printing and dyeing wastewater can be efficiently treated. And 5, the lignin degradation capacity is outstanding, the degradation rates within 1 hour, 2 hours and 24 hours are 22%, 25% and 58% respectively, and pulping bleaching and biomass recycling are facilitated.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a laccase and its applications. Background Technology

[0002] 1. Overview of Laccase Lacase (EC 1.10.3.2) is a copper-containing polyphenol oxidase belonging to the blue polycopper oxidase family. It can catalyze the oxidation of various phenolic and non-phenolic compounds while reducing molecular oxygen to water. Lacase has the characteristics of broad substrate spectrum, mild catalytic conditions, and environmental friendliness, and has important application value in bioremediation, textile industry, pulp bleaching, bioenergy and other fields.

[0003] 2. Problems with existing technologies (1) Insufficient activity: The reported activities of laccases are generally low, which is difficult to meet the needs of industrial applications; (2) Poor stability: Most laccases have limited tolerance to temperature and pH, which limits their application range; (3) High production cost: The discovery of highly active laccase mainly relies on traditional screening methods, which are inefficient and costly.

[0004] In the prior art, Huang et al. (2021) from Shandong University reported a method derived from the gut of termites. Bacillus stratosphericus The laccase BstCotA exhibits good thermostability and alkali resistance, with a specific activity of 554.1 U / mg. This enzyme is a representative of high-activity natural laccases, but there is still room for further improvement. Furthermore, patent application CN116064605A discloses a laccase, its gene, and its applications. The laccase gene of this invention was developed using *Trichoderma discolor*. Cerrena unicolor The laccase gene was obtained after codon optimization. The laccase gene provided by this invention, through recombinant expression via transformation of Aspergillus niger, can achieve large-scale industrial production of laccase with high enzyme activity, with the highest enzyme activity reaching 39 U / ml in shake flask culture. Summary of the Invention

[0005] This invention provides a novel laccase with higher activity than reported in existing literature, and the enzyme activity, pH stability and thermal stability are all significantly improved.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: First, this invention utilizes database mining to rapidly obtain highly active laccase candidate proteins. To verify its function and enzymatic properties, the laccase was synthesized and fused downstream of a His tag, recombinantly expressed in *E. coli*, and then subjected to Ni-NTA affinity chromatography to obtain the laccase candidate protein Lac-11.

[0007] The amino acid sequence of this laccase candidate protein is shown in SEQ ID NO.1. A deep search for distant homologous proteins using a protein language model (PLM) revealed that Lac-11 has a sequence similarity of 53.19% with BstCotA and 71.51% and 72.59% similarity to the most similar protein in the NCBI protein database, respectively, demonstrating excellent novelty.

[0008] On the other hand, the present invention also provides a nucleic acid molecule encoding the laccase.

[0009] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0010] In another aspect, the present invention also provides a carrier comprising the nucleic acid molecule.

[0011] Furthermore, in some embodiments of the present invention, the above-mentioned vector is a cloning vector or an expression vector; more preferably, it is a vector for use in bacteria, such as Escherichia coli (E. coli). E. coli The recombinant expression vector is pRSFDuet1. Preferably, in some embodiments of the present invention, the expression vector is pRSFDuet1.

[0012] In another aspect, the present invention also provides a host cell comprising the carrier.

[0013] This invention also provides the application of the laccase, the nucleic acid molecule, the carrier, or the host cell in an oxidizing substrate. The substrate includes the non-phenolic substrate ABTS (2,2'-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid)).

[0014] This invention explored the enzyme activity of Lac-11 on ABTS substrates. The results showed that Lac-11 had better enzyme activity on ABTS substrates, with an activity nearly 50% higher than that of laccase BstCotA at 37°C and pH 5.0.

[0015] This invention explored the optimal pH and temperature of Lac-11, and found that the optimal pH of Lac-11 is 4.0. After maintaining the enzyme activity of Lac-11 in an environment of pH 5.0-8.0 for 1 hour, the enzyme activity retention rate of Lac-11 is higher than that of BstCotA. The optimal temperature is 70℃. After maintaining the enzyme activity of Lac-11 in an environment of 50-80℃ for 1 hour, the enzyme activity retention rate of Lac-11 is higher than that of BstCotA.

[0016] This invention also provides the application of the laccase, the nucleic acid molecule, the carrier, or the host cell in lignin degradation. Lac-11 achieved lignin degradation rates of 22% and 25% at 1 hour and 2 hours, respectively, higher than BstCotA (19% and 22%), and a lignin degradation rate of 58% at 24 hours, significantly higher than BstCotA (40%).

[0017] The present invention also provides the application of the laccase, the nucleic acid molecule, the carrier, or the host cell in the decolorization of synthetic dyes, wherein the synthetic dye is selected from at least one of amino black 10B, crystal violet, Congo red, neutral red, alizarin red, and methyl orange.

[0018] Preferably, the application is implemented in a dye wastewater treatment system.

[0019] As an example, this invention explored the decolorization effect of Lac-11 on synthetic dyes, finding that Lac-11 had a decolorization effect on both Amine Black 10B and Crystal Violet. Specifically, after 3 hours, the decolorization rate of Amine Black 10B with Lac-11 was approximately 31%, higher than BstCotA (approximately 28%); the decolorization rate of Crystal Violet was approximately 37%, higher than BstCotA (approximately 32%). In conclusion, Lac-11 has a strong decolorization ability for a variety of dyes and can efficiently treat dyeing and printing wastewater, showing great promise for application in dye wastewater treatment.

[0020] This invention obtains a highly active laccase, Lac-11, through database mining. Compared with the existing highly active laccase BstCotA, it has the following advantages: 1. Significantly enhanced enzyme activity: enzyme activity increased by nearly 50% under conditions of 37℃ and pH 5.0, reducing the cost of industrial enzymes.

[0021] 2. It has stronger pH stability, higher enzyme activity retention rate in the pH range of 5.0-8.0, and can adapt to a wider range of acid and alkaline environments.

[0022] 3. It has better thermal stability, with significantly higher stability than the control enzyme in the medium-high temperature range of 50-80℃, making it suitable for industrial high-temperature processes.

[0023] 4. It has higher dye decolorization efficiency, with decolorization rates of 31% and 37% for Amine Black 10B and Crystal Violet respectively, and can effectively treat printing and dyeing wastewater.

[0024] 5. It has outstanding lignin degradation ability, with degradation rates of 22%, 25%, and 58% after 1 hour, 2 hours, and 24 hours, respectively, which is beneficial for pulping, bleaching, and biomass resource utilization. Attached Figure Description

[0025] Figure 1The image shows the recombinant expression plasmid His-Lac-11-pRSFDuet1 for Lac-11.

[0026] Figure 2 The graph shows the enzyme activity assay results of Lac-11 compared to BstCotA; *** indicates P<0.001.

[0027] Figure 3 The graph shows the enzyme activity assay results of Lac-11 at different pH values.

[0028] Figure 4 The graph shows the enzyme activity assay results of Lac-11 at different temperatures according to the present invention.

[0029] Figure 5 The image shows the decolorization effect of Lac-11 on Amino Black 10B and Crystal Violet; ns indicates P>0.05.

[0030] Figure 6 The graph shows the degradation rate of alkali lignin by Lac-11; ns indicates P>0.05, * indicates P<0.05, and *** indicates P<0.001. Detailed Implementation

[0031] The main experimental materials used in the following examples were sourced from: Escherichia coli host strain E. coli BL21(DE3) was purchased from Beijing Qingke Biotechnology Co., Ltd., and kanamycin was purchased from Dalian Baosheng Biotechnology Co., Ltd.; isopropyl-β-D-thiogalactopyranoside (IPTG) was a product of Promega; alkali lignin was purchased from Sigma; BstCotA laccase was synthesized and purified by gene design based on the codon preference of E. coli (the optimized amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4). Reagents related to protein purification and enzyme activity assay were all purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0032] Example 1: Preparation of His-Lac-11-pRSFDuet1 1. This invention discovers a laccase candidate protein, with the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2. To verify its function and enzymatic properties, the laccase was fused downstream of a His tag via gene synthesis. A plasmid (e.g., [example of plasmid]) was synthesized by GenScript Biotech Inc. Figure 1 As shown in the figure, the plasmid was transformed into BL21(DE3) Escherichia coli competent cells by heat shock; the transformed cells were plated on LB plates containing 50 μg / mL kanamycin, cultured overnight, and positive clones were selected for further verification. 2. Expanded culture: The selected positive clones were inoculated into LB liquid medium containing antibiotics for expanded culture; cultured at 37℃ and 220 rpm until the logarithmic growth phase; 3. Induction of expression: When the OD value of the bacterial culture reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 0.5mM, incubate at 16℃ and 220 rpm for 20 hours, add 0.5 mM CuSO4 to promote the correct folding of laccase, and let stand at 16℃ for 6 hours. 4. Centrifuge the induced bacterial culture, collect the E. coli precipitate, lyse the bacteria, release intracellular proteins, and obtain a protein solution; 5. Load the lysed protein solution onto a pre-equilibrated Ni-NTA affinity chromatography column. Utilize the specific binding of the His tag to Ni ions, and elute with a gradient of buffers containing different concentrations of imidazole. Collect the eluent containing the target protein to obtain laccase Lac-11.

[0033] Example 2: Enzyme activity assay of Lac-11 Using ABTS as a substrate, the cationic free radicals (ABTS•) generated after ABTS oxidation were determined. + The rate of increase of absorbance at 420 nm was determined kinetically in a multi-functional microplate reader, and the change in absorbance at 420 nm within 3 minutes was measured. The reaction system is shown in Table 1.

[0034] Table 1 The results are as follows Figure 2 As shown, Lac-11 significantly enhances enzyme activity, increasing by nearly 50% at 37°C and pH 5.0, which can reduce the cost of industrial enzymes.

[0035] Example 3: Characterization of optimal pH and pH stability of Lac-11 Using ABTS as a substrate, the optimal pH of Lac-11 was determined at 37°C in a buffer system with a final concentration of 50 mM citrate-phosphate (pH 3.0–8.0). To assess pH stability, enzyme samples were diluted 20-fold with citrate-phosphate buffer at 1.0 intervals from 3.0 to 8.0, incubated at 25°C for 60 minutes, and then residual activity was measured under standard conditions. All assays were repeated at least three times.

[0036] The results are as follows Figure 3 The results showed that the optimal pH of Lac-11 was 4.0, consistent with that of BstCotA; pH stability results showed that after being maintained in an environment of pH 5.0-8.0 for 1 hour, the enzyme activity retention rate of Lac-11 was higher than that of BstCotA (Table 2).

[0037] Table 2 Example 4: Characterization of Lac-11's optimal temperature and temperature stability Using ABTS as a substrate, the enzyme activity was measured in a 50 mM citrate-phosphate buffer system (pH 5.0) at 10°C intervals within the range of 40–90°C. To assess temperature stability, appropriately diluted enzymes were incubated at 10°C intervals within the range of 40–90°C for 60 minutes, and then the residual activity was measured under standard conditions. All measurements were repeated at least three times.

[0038] The results are as follows Figure 4 The results showed that the optimal temperature for Lac-11 was 70℃; the temperature stability results showed that after being maintained in an environment of 50-80℃ for 1 hour, the enzyme activity retention rate of Lac-11 was higher than that of BstCotA (Table 3).

[0039] Table 3 Example 5: Application of Lac-11 in dye decolorization Using the triarylmethane dye crystal violet and the diazo dye amino black 10B as decolorization substrates, a 500 μl reaction system included 50 mg / L dye, 1 μg laccase, 10 μM ABTS as a mediator, and 50 mM pH 5.0 sodium malonate buffer. After standing in a 50℃ water bath for 3 hours, the changes in absorbance before and after decolorization were measured by ultraviolet spectrophotometry at 589 nm and 618 nm.

[0040] The results showed that after 3 hours, the decolorization rate of Lac-11 for Amino Black 10B was approximately 31%, higher than that of BstCotA (approximately 28%); the decolorization rate of Crystal Violet was approximately 37%, higher than that of BstCotA (approximately 32%). Figure 5 (As shown).

[0041] Example 6: Application of Lac-11 in lignin degradation Alkali lignin was used as the substrate for lignin degradation. A 10 mg / mL alkali lignin solution was prepared. A 2 mL reaction system included 1 mg / mL alkali lignin, 1 μg laccase, 2 mM ABTS as a mediator, and 50 mM pH 5.0 sodium malonate buffer. A control group was used without alkali lignin, and an untreated group was used without enzyme and ABTS. Samples from different reaction systems were placed in a shaker at 50℃ and 180 rpm for 24 hours. Samples were taken hourly for analysis. After shaking, 500 μL of the solution was dissolved in an equal volume of DMSO, diluted 10-fold, and the absorbance at 280 nm was measured using a UV spectrophotometer.

[0042] Alkali lignin solutions with concentrations of 10, 20, 30, 40, and 50 mg / L were prepared, and their absorbance at 280 nm was measured using a UV spectrophotometer. The standard curve equation for alkali lignin was calculated as: y = 0.0166x - 0.011 (R0). 2 =0.9991), based on this, the concentration of alkali lignin can be obtained from the absorbance, and the degradation rate of alkali lignin can be calculated. The calculation formula is as follows: Alkali lignin degradation rate = (Alkali lignin concentration in untreated group - Alkali lignin concentration in treated group) / Alkali lignin concentration in untreated group × 100%.

[0043] The results are as follows Figure 6 The results showed that Lac-11 had lignin degradation rates of 22% and 25% at 1 hour and 2 hours, respectively, which were higher than those of BstCotA (19% and 22%); the lignin degradation rate at 24 hours was 58%, which was significantly higher than that of BstCotA (40%).

Claims

1. A laccase, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The nucleic acid molecule encoding the laccase of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A vector comprising the nucleic acid molecule of claim 2 or 3.

5. A host cell comprising the vector of claim 4.

6. The application of the laccase of claim 1, the nucleic acid molecule of claim 2 or 3, the carrier of claim 4, or the host cell of claim 5 in lignin degradation.

7. The application of the laccase of claim 1, the nucleic acid molecule of claim 2 or 3, the carrier of claim 4, or the host cell of claim 5 in the decolorization of synthetic dyes, wherein the synthetic dye is selected from at least one of amino black 10B, crystal violet, Congo red, neutral red, alizarin red, and methyl orange.

8. The application according to claim 7, characterized in that, The application is implemented in a dye wastewater treatment system.

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