A laccase mutant and its application in treating desizing wastewater

Abstract

The present invention discloses a laccase mutant and its application in treating desizing wastewater. The present invention uses wild-type laccase as the parent and obtains a high-activity mutant through multiple groups of mutations. The temperature of the concentrated desizing wastewater to be treated is 15-50°C, and the pH is 4-7, with broad applicable conditions. It has been verified that the laccase mutant of the present invention has an enzyme activity of more than 10 U / mg under the conditions of 25°C and pH 6.0 (common conditions for concentrated desizing wastewater treatment). Even under relatively extreme conditions of pH 4.0 or 7.0 and reaction temperature of 15°C or 50°C, the laccase variant of the present invention can still maintain an enzyme activity of more than 5.5 U / mg. Therefore, the laccase mutant of the present invention can be used for large-scale treatment of desizing wastewater with a temperature of 15-50°C and a pH of 4-7. Compared with the wild-type laccase with relatively low enzyme activity, the enzyme activity of the laccase mutant obtained in the present invention is significantly improved, with a maximum increase of 10 times, significantly reducing the wastewater treatment cost.

Description

Technical Field

[0001] The invention relates to the fields of bioengineering and desizing wastewater treatment, and in particular to a laccase mutant and application thereof in treating desizing wastewater. Background Art

[0002] Desizing wastewater is a type of industrial wastewater generated by the textile industry during the desizing process of fabrics. Desizing refers to the removal of sizing materials attached to the surface of fabrics. The main components of these sizing materials include starch, cellulose and synthetic polymers, so that the fabrics can be smoothly processed in subsequent dyeing, printing or finishing processes. However, desizing wastewater usually contains a large amount of organic pollutants, and these pollutants have poor biodegradability, so desizing wastewater seriously pollutes water bodies.

[0003] Traditional water treatment methods, such as physical adsorption and chemical precipitation, often have limited effects in treating desizing wastewater, and it is difficult to completely remove organic pollutants in the wastewater. In recent years, the application of laccase in purifying desizing wastewater has gradually received attention. Laccase can degrade a variety of pollutants in desizing wastewater, especially for starch substances. Laccase catalyzes oxidation reactions and converts them into smaller molecules or highly biodegradable products such as aldehydes and ketones. These products can be further decomposed by microorganisms, thereby effectively reducing the toxicity and harmfulness of wastewater.

[0004] In addition, desizing wastewater may contain some phenolic substances, which may be degradation products of certain dyes and surfactants. Laccase can convert phenolic substances into non-toxic quinone substances or other products that can be further degraded through oxidation reactions, thereby effectively removing toxic pollutants in water. At the same time, laccase can also degrade dyes or dyeing auxiliaries, especially for dyes with aromatic ring structures. Laccase oxidizes the aromatic structure of the dye molecule, causing it to break or convert it into a relatively non-toxic product, thereby achieving the purpose of decolorization and degradation.

[0005] Laccase shows high efficiency in decomposing complex organic matter, especially phenols, starches and dyes, and can significantly reduce the concentration of organic pollutants in water. As a biocatalyst, laccase has the advantages of mild reaction, non-toxicity and no secondary pollution. It meets the requirements of green chemistry and sustainable development and is an environmentally friendly wastewater treatment technology. Laccase can be used in combination with other water treatment methods such as physical adsorption, chemical precipitation, membrane filtration, etc., to enhance the effect of wastewater treatment and reduce the use of chemical agents through synergistic effects.

[0006] However, there are some problems with the current application of laccase. The enzyme activity of laccase is relatively low. When treating desizing wastewater, a large amount of laccase needs to be added, which leads to high treatment costs. Therefore, how to improve the enzyme activity of laccase is a technical problem that needs to be solved urgently. Summary of the invention

[0007] In view of the technical problems existing in the prior art, such as the low enzyme activity of laccase and the high cost of wastewater treatment, the present invention provides a laccase mutant and its application in treating desizing wastewater. By performing multiple groups of mutations on wild-type laccase to obtain a high-enzyme-activity mutant, and then using it to treat desizing wastewater, the treatment cost can be significantly reduced.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A laccase mutant, with wild-type laccase as the parent, undergoes the following mutations: L463F + E471Q + A483P + R596H + K240M + N249Y + Q406L + K416N.

[0010] Furthermore, the amino acid sequence of the wild-type laccase is as shown in SEQ ID NO.1, and the amino acid sequence of the wild-type laccase is derived from the protein database NCBI, GenBank: KAG7410754.1.

[0011] Furthermore, the wild-type laccase is derived from Fusarium oxysporum f. sp. rapae.

[0012] Furthermore, it also includes a mutation set: Q44H + H97G + D399H.

[0013] Furthermore, it also includes a mutation set: N53K + N174T + N183P.

[0014] Furthermore, it also includes a mutation set: N53S + K361Q.

[0015] Furthermore, it also includes a mutation set: L69M + H83R + L438Q + V450L.

[0016] Furthermore, it also includes a mutation set: Q44H + N174T + D399H + H83Q + S89P + T102N + K381Q.

[0017] The object of the present invention also includes providing the following technical solutions:

[0018] A recombinant genetic material of laccase, which is DNA or RNA capable of expressing the above laccase mutant.

[0019] A recombinant plasmid of laccase, including the above recombinant genetic material of laccase mutant, such as inserting the nucleotide sequence of the target gene into the plasmid pET28a(+) through DNA recombination technology.

[0020] A recombinant cell of a laccase mutant, comprising a recombinant plasmid capable of expressing the above laccase mutant.

[0021] The application of the above laccase mutant in treating desizing wastewater is specifically as follows: The above laccase mutant is put into desizing wastewater, the temperature of the desizing wastewater is 15 - 50 °C, the pH is 4 - 7, and the laccase mutant can oxidatively degrade organic pollutants well.

[0022] Term explanation or definition:

[0023] "Recombinant gene": refers to the DNA that can transcribe and express laccase (RNA is usually not used as the direct expression form of a gene, but participates in protein synthesis as a transcription product of DNA). Generally, the synthesis of a recombinant gene starts in vitro and can be synthesized by the solid-phase phosphoramidite triester method, the TdT biosynthesis method, or other suitable techniques known in the art. After obtaining the template sequence, it can be amplified by PCR amplification technology or other suitable techniques known in the art. After constructing a recombinant strain, further large-scale amplification of the recombinant gene can be achieved by culturing the strain. In addition, the recombinant gene may also contain residual sequences of restriction enzyme cleavage sites, other accessory elements (such as control elements, including promoters, etc.), labeling substances (such as fluorescent labels, etc.), and other sequences that do not affect the expression of the target gene.

[0024] "Cloning scar": refers to the additional sequences that may be introduced into the gene expression region during gene cloning due to the binding of the inserted foreign DNA fragment to the vector. These additional sequences may include promoter sequences, ribosome binding sites (RBS), and signal peptide sequences. The promoter sequence is responsible for initiating the transcription of mRNA, the RBS is responsible for attracting ribosomes and initiating the translation process, and the signal peptide sequence helps in the transport and localization of proteins. The mature protein is usually cloned after the signal peptide and is thus cleaved from the signal peptide by signal peptidase when passing through the membrane. When cloning a construct after the signal peptide, restriction enzymes usually require specific sequences to cut DNA, which leaves a cloning scar after the signal peptide sequence.

[0025] "Signal peptide": usually a short peptide 16 - 30 amino acids long, present at the N-terminus of most newly synthesized proteins that are destined to enter the secretory pathway. The signal peptide is also known as a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence, or leader peptide. The signal peptide is usually cleaved from the protein by signal peptidase.

[0026] "Signal peptide cleavage site": Refers to the specific dipeptide position where signal peptidase cleaves the signal peptide from the mature protein. In most (but not all) cases, this dipeptide is Ala-Ala. The signal peptide cleavage site can be predicted by algorithms such as SignalP 4.1, which is available online at http: / / www.cbs.dtu.dk / services / SignalP / (Center for Biological Sequence Analysis, Technical University of Denmark).

[0027] "Promoter": Refers to the DNA region near the transcription start site of a gene, usually located upstream on the same strand (the 5' region pointing to the sense strand), which is responsible for initiating the transcription process of a specific gene and transcribing the gene information into messenger RNA (mRNA). According to its regulatory characteristics, promoters can be divided into two types: (1) Inducible promoters, which are operably linked to specific genes and whose expression can be activated by the presence of inducer substances. The expression of this type of promoter is usually strictly regulated and induced through specific signaling pathways or environmental conditions; (2) Constitutive promoters, which are not regulated by any inducer substances and can continuously drive the transcription of genes regardless of whether the environmental conditions contain inducers. Constitutive promoters maintain the basic gene expression level in cells and are crucial for many biological processes.

[0028] The abbreviation "RBS": Refers to the ribosome-binding site, or the binding site of the ribosome. This is the nucleotide sequence upstream of the start codon of the mRNA transcript, which is responsible for recruiting ribosomes during the initiation process of protein translation.

[0029] "Expression": Refers to the process of DNA being transcribed into messenger RNA (mRNA) and then translated into protein.

[0030] "Expression vector": Refers to a special DNA molecule that has the ability to incorporate and express heterologous polynucleotide fragments in host cells. Many prokaryotic and eukaryotic expression vectors are commercially available, and a suitable expression vector can be selected according to actual needs.

[0031] "Chassis cell": refers to a host vector used to express the DNA involved in the present invention. The host can include any organism capable of containing and expressing the nucleic acid or gene involved in the present invention, but is not limited thereto. Chassis cells can be prokaryotes or eukaryotes, single-celled or multi-celled, including mammalian cells, plant cells, fungi, etc. According to the prior art, those skilled in the art can achieve the heterologous expression of the recombinant DNA of the present invention in different disclosed chassis cells by adjusting parameters through a limited number of experiments. The chassis cells can be selected from at least one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansenula anomala, Candida spp., Rhodotorula spp., Bacillus spp., Escherichia spp., Salmonella spp., Clostridium spp., Streptomyces spp., Staphylococcus spp., Neisseria spp., Shigella spp. The present invention only lists the types of chassis cells and does not constitute a limitation on the types of chassis cells. The chassis cell is preferably Escherichia coli, and suitable Escherichia coli strains (including many other strains) include BL21(DE3), C600, DH5αF′, 1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, χ1776, XL1-Blue and Y1089 + etc. The above Escherichia coli strains are all commercially available strains.

[0032] "Identity": refers to the residues in two sequences being the same when the alignment is maximally corresponding. For example, if, when correctly aligned, the corresponding fragments of two sequences have the same residues at 5 out of 10 positions, it is said that the two sequences have 50% identity. Most bioinformatics programs report the percentage identity of the aligned sequence regions, which are usually not the entire molecule. If the alignment is long enough and contains enough identical residues, an expected value can be calculated, indicating that the same level in the alignment is unlikely to occur randomly.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The present invention obtains a high-activity mutant through multiple groups of mutations. Compared with the wild-type laccase with lower enzyme activity, the enzyme activity of the laccase mutant of the present invention is significantly improved. The desizing wastewater to be treated can adapt to a wide range of environmental conditions with a temperature of 15~50°C and a pH of 4~7. It has been verified that the laccase mutant of the present invention has an enzyme activity of more than 10 U / mg under the environmental conditions of 25°C and a pH of 6.0 (a relatively common situation for centralized treatment of desizing wastewater). Even under extreme conditions of a pH of 4.0 or 7.0 and a reaction temperature of 15°C or 50°C, the laccase variant of the present invention can still maintain an enzyme activity of more than 5.5 U / mg. Therefore, the laccase mutant of the present invention can be used for large-scale treatment of desizing wastewater with a temperature of 15~50°C and a pH of 4~7.

[0035] In summary, compared with the wild-type laccase (enzyme activity 1.547 U / mg), the mutant of the present invention has a very significant increase in enzyme activity, with the highest increase of 10 times, significantly reducing the wastewater treatment cost. Detailed implementation manners

[0036] The present invention will be further described in detail below in conjunction with specific embodiments, but the present invention is not limited thereto.

[0037] Laccase mainly oxidizes substrates through redox reactions, using the catalytic action of copper ions in laccase to oxidize substrates. The specific principle can be divided into the following key points:

[0038] 1. Oxidizing substrates: Laccase catalyzes substrates to carry out redox reactions, using molecular oxygen (O2) as an oxidant to transfer electrons in the substrates (including starch, phenolic compounds, dyes, etc.) to molecular oxygen, reducing it to water (H2O). Some organic substances in the substrates will be converted into products with low toxicity or biodegradability;

[0039] 2. Participation of copper ions: The catalytic activity of laccase comes from the copper ions it contains, and copper ions play a key role in the active sites of laccase (usually the exchange of divalent copper and monovalent copper ions). In the oxidation reaction, copper ions participate in the transfer of electrons and the reduction of oxygen, promoting the oxidation of substrates;

[0040] 3. Generation of quinone compounds: For starch substances, laccase oxidizes them into aldehyde, ketone or quinone compounds. These oxidation products are more easily degraded by microorganisms compared to the original substrates, reducing the pollution load of wastewater;

[0041] 4. Generation of water: Under the catalysis of laccase, oxygen is reduced to water, ensuring the progress of the reaction. Laccase effectively removes organic pollutants in wastewater through a series of redox reactions.

[0042] Example 1 Preparation of laccase

[0043] (1) Construction of recombinant cells:

[0044] The nucleotide sequences of the target genes were synthesized by Beijing Tsingke Biotechnology Co., Ltd. and inserted into the expression vector through molecular cloning technology. Specifically, these sequences were cloned into the plasmid pET28a(+), generating the corresponding recombinant plasmids. Subsequently, these recombinant plasmids were introduced into Escherichia coli BL21(DE3)) cells through transformation, thus constructing Escherichia coli strains containing different plasmids, namely recombinant bacteria. It should be noted that the prior art can provide a variety of available plasmids and chassis cells, and only one specific scheme is provided in this example.

[0045] (2)Expression and purification of laccase:

[0046] Inoculate the recombinant bacteria into liquid LB medium (5 g yeast extract, 10 g peptone, 10 g), and culture them in a shaker at 37 °C (rotation speed 220 rpm) until the OD 600 value is about 5.0. Then add IPTG to a concentration of 200 μM and lower the temperature to 16 °C, and culture overnight. After overnight culture, centrifuge at 4000 g for 10 min to collect the bacterial cells, and resuspend them in 10% volume of buffer NTA (50 mM Tri-HCl, 300 mM NaCl, pH 8.0). Then, disrupt the bacterial cells by sonication, and then centrifuge at 12000 rpm for 1 h. Take the supernatant as the crude enzyme solution. There are various available methods for inducing the expression of recombinant bacteria in the prior art, and only one specific scheme is provided in this example.

[0047] (3)Perform affinity chromatography on the crude enzyme solution through Ni 2+ resin. Since the C-terminus of the expressed target protein carries a histidine tag, it can specifically bind to the resin. First, wash the resin with buffer NTA and imidazole buffer to remove unbound impurities, and then elute with buffer NTA to collect the eluate containing the target protein. Dialyze the obtained solution to remove imidazole. The cut-off molecular weight of the dialysis bag is 1 kDa. After dialysis, concentrate the solution until the protein concentration reaches 0.2 mg / mL, and then perform lyophilization to obtain laccase powder. According to the prior art, those skilled in the art can adjust the parameters through a limited number of experiments to achieve the purification of laccase, and specific descriptions are not provided here. It is worth noting that there are various available laccase purification methods in the prior art, and only one specific scheme is provided in this example.

[0048] Perform heterologous expression and purification on wild-type laccase (LAC-1, SEQ ID NO: 1) and laccase mutants (LAC-2 to LAC-7) according to the above laccase preparation method.

[0049] Example 2 Enzyme activity assay:

[0050] 1. Reaction system: In a total reaction volume of 10 mL, it contains 1.1 mol / L 3-ethylbenzothiazole-6-sulfonic acid (ABTS), 0.1 mol / L acetate buffer, 0.02 g / L laccase, and the system pH is 5.0.

[0051] 2. Methods for measuring the enzyme activity of different laccases:

[0052] The reaction was carried out according to the above reaction system, and the reaction temperature was 25°C. The wild-type laccase and the laccase mutant were respectively put into the same reaction system. ABTS was converted into ABTS radical under the catalysis of laccase. Since the absorption coefficient of ABTS radical at 420 nm was significantly higher than that of the substrate ABTS, the absorbance value gradually increased with the increase of the concentration of ABTS radical. Therefore, by detecting the change of OD value at 420 nm, the enzyme activity of wild-type laccase or laccase mutant could be determined.

[0053] Definition of enzyme activity: Under the above conditions (25°C, pH 5.0), the increase in absorbance value (△OD 420 / (mg·min)) caused by 1 mg of laccase oxidizing the substrate (ABTS) per minute. The enzyme activities of the wild-type laccase and the laccase variants measured are shown in Table 1:

[0054] Table 1 Enzyme activities of wild-type laccase and laccase variants

[0055]

[0056] As can be seen from Table 1, the enzyme activity of the wild-type laccase was relatively low, only 1.547 U / mg, while the enzyme activities of the laccase mutants obtained by multiple mutations in the present invention were significantly improved, all above 10 U / mg.

[0057] Example 3 Determination of enzyme activity of laccase mutant under different temperature conditions

[0058] The reaction was carried out according to the reaction system of Example 2, with pH 5.0. The reaction temperature was changed to 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C respectively. The laccase mutants were respectively put into the same reaction system, and the measurement results are shown in Tables 2 - 7:

[0059] Table 2 Enzyme activities of laccase variant LAC-2 at different temperatures

[0060]

[0061] Table 3 Enzyme activities of laccase variant LAC-3 at different temperatures

[0062]

[0063] Table 4 Enzyme activities of laccase variant LAC-4 at different temperatures

[0064]

[0065] Table 5 Enzyme activities of laccase variant LAC-5 at different temperatures

[0066]

[0067] Table 6 Enzyme Activity of Laccase Variant LAC-6 at Different Temperatures

[0068]

[0069] Table 7 Enzyme Activity of Laccase Variant LAC-7 at Different Temperatures

[0070]

[0071] As can be seen from Tables 2 to 7, laccase variants LAC-2 to LAC-7 can maintain relatively high enzyme activity in the environment of pH 5.0 and 15 - 50 °C.

[0072] Example 4 Determination of Enzyme Activity of Laccase Mutants under Different pH Conditions

[0073] The reaction was carried out according to the reaction system of Example 2 (adjusting the pH by replacing the buffer solution), the reaction temperature was 25 °C, and the pH was changed to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 respectively. The laccase mutants were respectively put into the same reaction system, and the measurement results are shown in Tables 8 - 13:

[0074] Table 8 Enzyme Activity of Laccase Variant LAC-2 at Different pH Values

[0075]

[0076] Table 9 Enzyme Activity of Laccase Variant LAC-3 at Different pH Values

[0077]

[0078] Table 10 Enzyme Activity of Laccase Variant LAC-4 at Different pH Values

[0079]

[0080] Table 11 Enzyme Activity of Laccase Variant LAC-5 at Different pH Values

[0081]

[0082] Table 12 Enzyme Activity of Laccase Variant LAC-6 at Different pH Values

[0083]

[0084] Table 13 Enzyme Activity of Laccase Variant LAC-7 at Different pH Values

[0085]

[0086] As can be seen from Tables 8 to 13, laccase variants LAC-2 to LAC-7 can maintain relatively high enzyme activity in an environment of 25°C and pH 4.0 to 7.0.

[0087] Example 5 Determination of Laccase Mutant Enzyme Activity under Different Temperature and pH Conditions

[0088] The reaction was carried out according to the reaction system of Example 2, with a pH of 4.0 or 7.0 and a reaction temperature of 15°C or 50°C. The laccase mutants were respectively added into the same reaction system, and the measurement results are shown in Table 14:

[0089] Table 14 Laccase Mutant Enzyme Activity under Different Temperature and pH Conditions

[0090]

[0091] As can be seen from Table 14, in an environment with a pH of 4.0 or 7.0 and a reaction temperature of 15°C or 50°C, laccase variants LAC-2 to LAC-7 can still maintain relatively high enzyme activity. Therefore, the laccase mutants of the present invention can be used to treat desizing wastewater in a relatively wide range of temperatures from 15 to 50°C and pH from 4 to 7.

Claims

1. A laccase mutant, characterized in that: The wild-type laccase with the amino acid sequence shown in SEQ ID NO. 1 was used as the parent, and the following mutations were performed: L463F+E471Q+A483P+R596H+K240M+N249Y+Q406L+K416N.

2. The laccase mutant according to claim 1, characterized in that Also included is the mutation set: Q44H+H97G+D399H.

3. The laccase mutant according to claim 1, characterized in that Also included is the mutation set: N53K+N174T+N183P.

4. The laccase mutant according to claim 1, characterized in that Also included is the mutation set: N53S+K361Q.

5. The laccase mutant according to claim 1, characterized in that Also included is the mutation set: L69M+H83R+L438Q+V450L.

6. The laccase mutant according to claim 1, characterized in that Also included is the mutation set: Q44H+N174T+D399H+H83Q+S89P+T102N+K381Q.

7. A recombinant genetic material of a laccase mutant, characterized in that: The recombinant genetic material is a DNA or RNA capable of expressing the laccase mutant according to any one of claims 1 to 6.

8. A recombinant plasmid of a laccase mutant, characterized in that: A recombinant genetic material comprising the laccase mutant according to claim 7.

9. A laccase mutant recombinant cell, characterized in that: The invention comprises a recombinant plasmid capable of expressing the laccase mutant according to claim 8.

10. Use of the laccase mutant according to any one of claims 1 to 6 in treating desizing wastewater, characterized in that: The laccase mutant is put into desizing wastewater for oxidative degradation, and the temperature of the desizing wastewater is 15-50° C. and the pH is 4-7.

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