Laccase bc-lac and its coding gene and application

Abstract

The application discloses laccase Bc-LAC from Botrytis cinerea, and an encoding gene and application thereof. The encoding gene of the laccase Bc-LAC is constructed into Pichia pastoris to obtain laccase Bc-LAC producing recombinant bacteria, the fermentation activity of a 30-liter fermentation tank reaches 15.83 U / mL, and the recombinant enzyme can effectively degrade lignin in rice straw and corn straw. The laccase Bc-LAC has the highest catalytic activity at pH 2.5, has good pH stability, has the highest catalytic activity at a temperature of 40 DEG C, and after being treated by metal ions (such as Cu 2+ ), the catalytic activity of the laccase Bc-LAC can be increased by about 10.6%. The laccase Bc-LAC can be widely used in papermaking, environmental protection, food and feed and other fields.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to laccase Bc-LAC, its encoding gene, and its applications. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass, with an estimated annual global production of 1×10⁻⁶. 8 Hundreds of millions of tons. Especially valuable are the lignocellulose wastes from the agricultural and forestry industries, which are low-cost and abundant. Lignocellulose is mainly composed of lignin, cellulose, and hemicellulose, and its complex structure makes it difficult to degrade and utilize. Lignin acts as a physical barrier, preventing the degradation of digestible cellulose and hemicellulose. Therefore, lignin removal can alter the structural characteristics of lignocellulose, increasing the contact between hydrolytic enzymes and cellulose and hemicellulose. Biological pretreatment is the most effective way to degrade lignin, with lignin-degrading enzymes such as peroxidase and laccase playing a key role in this process.

[0003] However, the yield of lignin-degrading enzymes in natural strains is low, making large-scale production economically impossible. Studies have shown that genetic engineering techniques are very useful for improving enzyme expression levels and stability.

[0004] Laccase (EC 1.10.3.2), also known as phenol oxidase or polycopper oxidase, utilizes molecular oxygen to catalyze the oxidation of various aromatic and non-aromatic substances, and is one of the main enzyme systems for degrading lignin. Laccases are widely distributed in prokaryotes, plants, fungi, and insects. Most laccases are glycoproteins with a molecular weight of approximately 60-80 kDa, an optimal pH that is slightly acidic, and an optimal temperature that is relatively low, generally between 26 and 50 °C. Laccases have a broad substrate spectrum and utilize only molecular oxygen as an electron acceptor without the need for peroxides, making them environmentally friendly. These characteristics have led to their widespread application in dye decolorization, pesticide detoxification, lignin degradation, food processing, and the pharmaceutical industry.

[0005] Laccase has a broad substrate range. It is estimated that laccase can catalyze up to 250 substrates, including phenols and their derivatives, aromatic amines and their derivatives, carboxylic acids and their derivatives, steroid hormones and biopigments, metal compounds, and other non-phenolic substrates. Laccase from different sources exhibits varying substrate specificity. Commonly used substrates for determining laccase activity include guaiacol, eugenol, and 2,2-adiazonium-bis(3-ethyl-benzothiazole-6-sulfonic acid) (ABTS). Laccase's broad substrate spectrum makes it a promising candidate for the degradation and utilization of lignocellulose. Summary of the Invention

[0006] The purpose of this invention is to provide a novel laccase Bc-LAC, its encoding gene, and its applications.

[0007] To achieve the objectives of this invention, in a first aspect, the invention provides laccase Bc-LAC, said laccase comprising or consisting of the following amino acid sequence:

[0008] i) An amino acid sequence from Botrytis cinerea as shown in SEQ ID NO:3; or

[0009] ii) The amino acid sequence obtained by attaching a tag to the N-terminus and / or C-terminus of i); or

[0010] iii) An enzyme with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of i) or ii).

[0011] Secondly, the present invention provides a gene encoding the laccase. Its nucleotide sequence is shown in SEQ ID NO:1, and its CDS sequence is shown in SEQ ID NO:2. The nucleotide sequence optimized according to the Pichia pastoris codon preference is shown in SEQ ID NO:4, and the encoded laccase protein does not contain a signal peptide.

[0012] Thirdly, the present invention provides biological materials containing the said gene, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0013] Fourthly, the present invention provides a recombinant bacterium that produces laccase Bc-LAC. The recombinant bacterium is constructed by codon optimization of the gene encoding laccase Bc-LAC, followed by introduction into yeast via plasmid or integration into the yeast chromosome via genetic engineering.

[0014] Preferably, the yeast is Pichia pastoris, more preferably Pichia pastoris X33.

[0015] Fifthly, the present invention provides a method for constructing a recombinant bacterium producing laccase Bc-LAC, the method comprising: constructing the gene encoding laccase Bc-LAC into the expression vector pPICZαA after codon optimization (SEQ ID NO:4), introducing the resulting recombinant vector into Pichia pastoris X33, and screening for positive transformants.

[0016] Furthermore, recombinant laccase was isolated and purified from the laccase-producing Bc-LAC recombinant bacteria.

[0017] In a sixth aspect, the present invention provides any of the following applications of the laccase, its encoding gene, biological material containing the gene, the recombinant bacteria, or the recombinant bacteria constructed according to the method:

[0018] 1) Used to prepare products with laccase activity;

[0019] 2) Used for lignin degradation.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] (I) The laccase-producing Bc-LAC recombinant bacteria provided by this invention has a fermentation activity of 15.83 U / mL in a 30-liter fermenter. The recombinant enzyme can effectively degrade lignin in rice straw and corn straw.

[0022] (II) Laccase Bc-LAC exhibits the highest catalytic activity and good pH stability at pH 2.5; its catalytic activity is also highest at 40℃; it reacts with metal ions (such as Cu) 2+ After treatment, the catalytic activity of laccase Bc-LAC can be increased by about 10.6%. Laccase Bc-LAC can be widely used in papermaking, environmental protection, food and feed industries. Attached Figure Description

[0023] Figure 1 This is an SDS-PAGE electrophoresis image of the supernatant from the shake-flask fermentation of the recombinant strain X33 / Bc-Lac in a preferred embodiment of the present invention. Wherein, M: protein molecular weight standard; I: fermentation supernatant of X33 / Bc-Lac.

[0024] Figure 2 The curves showing the wet weight of cells and the growth of enzyme activity in a 30L fermenter are shown in a preferred embodiment of the present invention.

[0025] Figure 3 The effect of pH value on the activity of laccase Bc-LAC in a preferred embodiment of the present invention.

[0026] Figure 4 The pH stability of laccase Bc-LAC in a preferred embodiment of the present invention.

[0027] Figure 5 The effect of temperature on the activity of laccase Bc-LAC is shown in a preferred embodiment of the present invention.

[0028] Figure 6 The temperature stability of laccase Bc-LAC in a preferred embodiment of the present invention.

[0029] Figure 7 The preferred embodiment of the present invention shows the degradation effect of laccase Bc-LAC treatment on lignin in rice straw and corn straw.

[0030] Figure 8These are scanning electron microscope (SEM) images of rice straw and corn straw treated with laccase Bc-LAC in a preferred embodiment of the present invention. Specifically: a. SEM image of untreated rice straw; b. SEM image of rice straw treated with laccase Bc-LAC; c. SEM image of untreated corn straw; d. SEM image of corn straw treated with laccase Bc-LAC. Detailed Implementation

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0032] Example 1: Cloning, optimization design, and synthesis of the mafic-YANG laccase gene Bc-lac from Botrytis cinerea.

[0033] Botrytis cinerea mafic-YANG was isolated and preserved by the Feed Industry Center Laboratory of the College of Animal Science and Technology, China Agricultural University. Primers Bclac3-F (5′-ATGAAGTTGTTCAATATTCTA-3′) and Bclac3-R (5′-TTAGACACCAGAATCGGT-3′) were designed based on the Botrytis cinerea laccase gene sequence reported in GenBank (NCBI accession number: MT707622.1). Using Botrytis cinerea genomic DNA as a template, the Bc-lac gene was amplified by PCR and ligated into the pGM-T cloning vector. This vector was then transformed into Escherichia coli Top10 competent cells to construct the recombinant cloning plasmid pGM-Bc-lac. Screening was performed using ampicillin and blue-white plate assays. Positive strains were selected, and after expansion culture, the recombinant plasmid pGM-Bc-lac was extracted and sequenced for verification. Bc-lac is 2107 bp in length (SEQ ID NO:1) and contains two intron sequences. The full-length Bc-lac CDS is 1992bp (SEQ ID NO:2), encoding 663aa (SEQ ID NO:3), where 1-19aa is the signal peptide sequence, and the predicted molecular weight of the mature protein is 69.1kDa.

[0034] Example 2: Codon optimization design and synthesis of the laccase gene Bc-lac

[0035] Without altering the amino acid sequence, the Bc-lac mRNA sequence was optimized according to the codon preference of Pichia pastoris. Low-frequency codons were replaced with high-frequency ones, and the secondary structure of the mRNA was adjusted to reduce the free energy for opening the secondary structure. Simultaneously, the leading signal peptide sequence (57 bp) was removed. The optimized gene Bc-lac-opt sequence is shown in SEQ ID NO:4, encoding 644aa, with a predicted molecular weight of 69.1 kDa. The optimized sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd., and restriction endonuclease EcoRI and XbaI restriction sites were added to the 5′ and 3′ ends, respectively. The resulting plasmid, pPICZαA, was obtained and stored in *E. coli* Top10. The resistance gene is Zeocin.

[0036] Example 3: Construction and shake-flask fermentation of Pichia pastoris strain expressing the laccase gene Bc-lac-opt.

[0037] E. coli containing the recombinant plasmid pPIC-Bc-lac-opt were streaked onto LB agar containing zeocin (25 μg / mL) and incubated at 37°C until single colonies appeared. A single colony was picked and inoculated into LB liquid medium containing zeocin (25 μg / mL) and incubated overnight at 37°C at 220 rpm. The recombinant plasmid was extracted using a plasmid extraction kit (Omega, USA). The recombinant plasmid pPIC-Bc-lac-opt was linearized with the restriction endonuclease Sac I and digested overnight at 37°C. After enzyme digestion, the plasmid was purified using a DNA purification kit (Omage, USA), and then electroporated (voltage 2.0 kV, capacitance 25 μF, resistance 200 Ω, electroporation time 5 ms) into Pichia pastoris X-33 competent cells. 100 μL of the plasmid was spread on YPDS solid medium containing Zeocin (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 1 mol / L sorbitol, 20 g / L agar, autoclaved at 115℃ for 15 min), and cultured upside down at 30℃. The resulting strain was Pichia pastoris X33 / Bc-lac engineered strain.

[0038] Single colonies of X33 / Bc-lac were picked and inoculated into 10 mL of YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, autoclaved at 115°C for 15 min) liquid medium containing Zeocin (100 μg / mL). The culture was incubated at 28°C and 220 rpm for approximately 24 h. 100 μL of the bacterial culture was then transferred to 50 mL of BMGY (10 g / L, 20 g / L peptone, 1.34% YNB, 4 × 10⁻⁶ ppm). -5The culture medium (1% biotin, 1% glycerol, 100 mM pH 6.0 phosphate buffer) was placed in a 500 mL shake flask and cultured at 28 °C and 250 rpm with shaking. After 24 h, the culture was centrifuged at 5,000 rpm and 4 °C for 5 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in 50 mL of BMMY medium and placed in a 500 mL Erlenmeyer flask. The culture was then incubated at 28 °C and 220 rpm, with anhydrous methanol added to the medium every 12 h to induce expression. After 96 h, the bacterial culture was centrifuged at 5,000 rpm and 4 °C for 5 min, and the supernatant was collected. The laccase activity was measured to be 0.26 U / mL using ABTS as a substrate at pH 3.0 and 30 °C. The supernatant was collected and analyzed by 12% SDS-PAGE electrophoresis, which showed a clear protein band of approximately 70 kDa. Figure 1 ).

[0039] The method for determining laccase activity is as follows: Prepare 1 mmol / L ABTS solution using citrate-disodium hydrogen phosphate buffer. Mix 50 μL of enzyme solution with 950 μL of citrate-disodium hydrogen phosphate buffer. Separately, place 1 mL of ABTS solution into another centrifuge tube and preheat both solutions to 30°C for 3 min. Then, mix the two solutions in a centrifuge tube. Incubate the mixture at 30°C for 10 min and measure the change in absorbance at 420 nm using a microplate reader. The definition of a laccase activity unit (U) is: the amount of enzyme required to oxidize 1 μmol of ABTS per minute under conditions of pH 2.5 and 40°C.

[0040] Example 4: High-density fermentation of X33 / Bc-lac in a 30L fermenter

[0041] The Pichia pastoris yeast strain X33 / Bc-lac expressing laccase Bc-Lac obtained in Example 3 was selected and inoculated into 10 mL of YPD medium. After culturing at 220 rpm and 28°C for 24 h, 1 mL of the bacterial culture was transferred to a 500 mL Erlenmeyer flask containing 100 mL of YPD liquid medium and cultured at 220 rpm and 28°C. 600When the culture reaches stages 2-6, 0.6L of bacterial culture is added to a 30L fermenter containing 12L of sterile fermentation basal medium (containing 50g glucose, 5g KH2PO4, CaSO4·2H2O, 18.2g K2SO4, 14.9g MgSO4·7H2O, 50g NH4H2PO4, 1.5g KOH and 4.37mL PTM1 salt per liter; the PTM1 salt formula is: 6g CuSO4·5H2O, 0.09g KI, 3g MnSO4·H2O, 0.2g Na2MoO4·2H2O, 0.02g H3BO3, 0.5g CoCl2·6H2O, 20g ZnCl2, 65g FeSO4·7H2O, 5mL 98.3% concentrated sulfuric acid, and 100mL 0.2% biotin per liter) for staged culture. The initial conditions were: temperature 28.5℃, pH adjusted to 5.0 with ammonia, and relative dissolved oxygen maintained above 20% during fermentation by adjusting the turbine speed and air flow rate. At 26.3 hours of fermentation, the basic carbon source, glucose, was depleted, and 50% glycerol (w:v) was introduced as the basic carbon source for feeding. The feeding rate was controlled according to changes in dissolved oxygen levels to maintain a dissolved oxygen level above 20%. When the cell wet weight reached 200.4 g / L, glycerol feeding was stopped, and the methanol induction phase began after the cells had consumed the remaining glycerol. Relative dissolved oxygen was maintained above 20% by adjusting the turbine speed, air flow rate, and methanol feeding rate. Cell wet weight and laccase activity were measured every 12 hours. Figure 2 When the cell wet weight and enzyme activity remain stable or show a downward trend, fermentation is stopped. At this time, the cell wet weight is 320.7 g / L and the laccase activity is 15.83 U / mL.

[0042] Example 5: Analysis of the enzymatic properties of recombinant laccase Bc-LAC

[0043] 1. Optimal catalytic pH and pH stability

[0044] Citrate-disodium hydrogen phosphate buffer solutions were prepared with pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The enzyme solution and substrate were diluted appropriately using these buffer solutions at different pH values, and enzyme activity was measured at 40°C to investigate the effect of different pH values ​​on Bc-LAC enzyme activity. Figure 3 As shown, Bc-LAC exhibits the highest catalytic activity at pH 2.5, maintains above 40% under pH conditions of 3.0–5.0, and almost loses its catalytic activity at pH conditions of 6.0–8.0.

[0045] After appropriate dilution, the enzyme solution was placed in citrate-disodium hydrogen phosphate buffer at pH values ​​of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, and incubated at room temperature for 30 min. The residual enzyme activity was then measured under optimal conditions. Figure 4 As shown, after treatment at room temperature for 30 min under different pH conditions, the enzyme activity of Bc-LAC remained almost unchanged, indicating good pH stability.

[0046] 2. Optimal catalytic temperature and temperature stability

[0047] A 1 mM ABTS solution was prepared using a citrate-disodium hydrogen phosphate buffer solution (pH 2.5), and the enzyme solution was appropriately diluted. The enzyme activity of Bc-LAC was measured between 10 and 80 °C to determine the effect of temperature on enzyme activity. Figure 5 As shown, within the temperature range of 10–40℃, the catalytic activity of Bc-LAC gradually increases with increasing temperature. The catalytic activity of laccase Bc-LAC is highest at 40℃; within the temperature range of 40–60℃, the enzyme activity of Bc-LAC changes relatively little; when the temperature exceeds 60℃, the activity decreases rapidly, and at 80℃, it almost loses its activity.

[0048] In a citrate-disodium hydrogen phosphate buffer solution at pH 2.5, Bc-LAC was incubated at 60°C and 65°C for 180 min, and at 70°C and 75°C for 120 min and 40 min, respectively, with samples taken at regular intervals. Figure 6 As shown, the enzyme activity of Bc-LAC was measured at 40℃ and pH 2.5, and the residual enzyme activity was calculated. Bc-LAC exhibited good temperature stability at 60℃ and 65℃, with residual enzyme activities of 83% and 34% after 180 min of treatment, respectively. However, after 60 min of treatment at 70℃, the residual enzyme activity was only 40%, indicating that Bc-LAC has good temperature stability.

[0049] 3. Stability of metal ions

[0050] Solutions of MnSO4, (NH4)2SO4, FeSO4, KCl, ZnSO4, CoSO4, CaCl2, NaCl, MgCl2, and CuSO4 were prepared using a citrate-disodium hydrogen phosphate buffer solution at pH 2.5. Appropriately diluted enzyme solutions were mixed with metal ion solutions to achieve a final metal ion concentration of 10 mmol / L. After treatment at room temperature for 1 h, residual enzyme activity was measured at pH 2.5 and 40 °C. A control was prepared by mixing the pH 2.5 buffer solution and enzyme solution without added metal ions in the same proportion (relative activity was 100%). The results are shown in Table 1. Except for Fe... 2+ and Cu 2+Apart from Fe, other metal ions do not affect the activity of Bc-LAC. 2+ After treatment, the activity of Bc-LAC was only 1.7%, while Cu... 2+ The Bc-LAC activity increased by 10.6% after treatment.

[0051] Table 1. Effects of metal ions on laccase Bc-LAC activity

[0052]

[0053] Example 6: Study on the degradation of lignin in rice and corn straw by laccase Bc-LAC

[0054] 1. Effect of laccase Bc-LAC on lignin degradation efficiency

[0055] Substrate preparation: Wash rice straw and corn straw with distilled water, dry them at 70℃, crush them and pass them through a 40-mesh sieve for later use.

[0056] Substrate (crushed rice straw and corn straw) and laccase were placed separately in Erlenmeyer flasks containing citrate-disodium hydrogen phosphate buffer (pH 3.0) at a ratio of 1:20 (w / v) and treated in a shaker at 150 rpm and 30°C. The amount of laccase used was 30 U per gram of substrate, with heat-inactivated laccase as a control (three replicates per group). Enzymatic hydrolysis was performed in a shaker for different times, and the lignin content was determined. The specific method (refer to Zhang Hongman et al., Analytical Laboratory, 2010, 29(11):15-18.) is as follows: Weigh 0.5g of sample (m), place it in a fiber bag with a mass of m1, and treat it with 72% sulfuric acid (10-15mL per sample) at 30℃ for 1h; add distilled water to dilute the sulfuric acid to 4%, and treat it in a high-pressure steam sterilizer at 121℃ for 45min; wash it repeatedly with hot water (about 70℃) more than three times until there is no residual acid; dry it at 105℃ to constant weight and weigh it (m2); ashing it at 550℃ and measuring the ash weight m3.

[0057] Lignin degradation rate = (m2 - m3 - m1) / m × 100%

[0058] The results are as follows Figure 7 As shown, the delignification rate of laccase Bc-LAC increases with time, reaching its highest level at 32 hours. At this time, the delignification rates of rice straw and corn straw were 29.1% and 24.9%, respectively.

[0059] 2. Effects of laccase treatment on the surface structure of rice straw and corn straw

[0060] Untreated and treated lignocellulose samples (rice straw, corn straw) were dried in an electrically heated drying oven, and their surface morphology was characterized using a scanning electron microscope (S-3400N, Hitachi, Japan). The samples were gold-plated before analysis, and images were taken at 1000x magnification. The scanning electron microscope allows for direct observation of changes in the physical surface structure of the materials. Figure 8 As can be seen from images a and c, untreated rice straw and corn straw have smooth, flat, and dense surfaces, with neat, regular fiber bundles and natural stretching. After treatment with laccase Bc-LAC ( Figure 8 In samples b and d), the overall surface integrity is partially damaged, resulting in a rough surface with numerous pores, a loose structure, a porous texture, and disordered fiber breakage. Laccase treatment increases the porosity and roughness of the lignocellulose biomass surface, which in turn increases the surface area, allowing for greater contact with hydrolytic enzymes and improving the conversion rate of lignocellulose.

[0061] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Laccase Bc-LAC, characterized in that, The laccase comes from Botrytis cinerea ( Botrytis cinerea The amino acid sequence is shown in SEQ ID NO:

3.

2. The gene encoding the laccase of claim 1.

3. The gene according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

4.

4. A biomaterial containing the gene of claim 2 or 3, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

5. A recombinant bacterium producing laccase Bc-LAC, characterized in that, The recombinant bacteria were constructed by codon optimization of the gene encoding laccase Bc-LAC, followed by introduction into yeast via plasmid or integration into the yeast chromosome through genetic engineering. The optimized gene sequence is shown in SEQ ID NO:

4.

6. The recombinant bacteria according to claim 5, characterized in that, The yeast is Pichia pastoris (Pichia pastoris) Pichia pastoris )X33.

7. A method for constructing a laccase-producing Bc-LAC recombinant bacterium, characterized in that, The method includes: codon optimization of the gene encoding laccase Bc-LAC, construction into the expression vector pPICZαA, introduction of the resulting recombinant vector into Pichia pastoris X33, and screening for positive transformants; The optimized gene sequence is shown in SEQ ID NO:

4.

8. Any of the following applications of the laccase of claim 1, the gene of claim 2 or 3, the biomaterial of claim 4, the recombinant bacteria of claim 5 or 6, or the recombinant bacteria constructed according to the method of claim 7: 1) Used to prepare products with laccase activity; 2) Used for lignin degradation.

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