Laccase mutant and application thereof in desizing wastewater treatment

Abstract

The invention discloses a laccase mutant and application thereof in desizing wastewater treatment. Wild laccase is used as a female parent, and a high-enzyme-activity mutant is obtained through multiple groups of mutations. The temperature of the desizing wastewater subjected to centralized treatment is 15-50 DEG C, the pH value is 4-7, and the application condition is wide. Through verification, the enzyme activity of the laccase mutant disclosed by the invention is more than 10U / mg under the environment that the temperature is 25 DEG C and the pH value is 6.0 (common conditions for centralized treatment of desizing wastewater). Even under the extreme conditions that the pH is 4.0 or 7.0 and the reaction temperature is 15 DEG C or 50 DEG C, the laccase variant can still keep the enzyme activity of 5.5 U / mg or above, and therefore the laccase variant can be used for treating desizing wastewater with the temperature being 15-50 DEG C and the pH being 4-7 in a large scale. Compared with wild type laccase with lower enzyme activity, the laccase mutant obtained by the invention has the advantages that the enzyme activity is obviously improved and can be improved by 10 times at most, and the wastewater treatment cost is obviously reduced.

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 in the prior art such as low enzyme activity of laccase and high cost of treating wastewater, the present invention provides a laccase mutant and its application in treating desizing wastewater. By subjecting the wild-type laccase to multiple groups of mutations to obtain mutants with high enzyme activity, which are then used to treat desizing wastewater, the treatment cost can be significantly reduced.

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

[0009] A laccase mutant, with the 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 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, a mutation set is also included: N53K+N174T+N183P.

[0014] Furthermore, a mutation set is also included: 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 purpose of the present invention also includes providing the following technical solutions:

[0018] The invention relates to a recombinant genetic material of laccase, which can express the DNA or RNA of the laccase mutant.

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

[0020] A laccase mutant recombinant cell comprises a recombinant plasmid capable of expressing the laccase mutant.

[0021] The application of the laccase mutant in treating desizing wastewater is specifically as follows: the laccase mutant is added to the desizing wastewater, the temperature of the desizing wastewater is 15-50°C, and the pH is 4-7. The laccase mutant can perform good oxidation and degradation of organic pollutants.

[0022] Explanation or definition of terms:

[0023] "Recombinant gene": refers to DNA that can transcribe and express laccase (RNA is usually not a direct expression form of the gene, but participates in protein synthesis as a transcription product of DNA). Usually, the synthesis of recombinant genes begins in vitro and can be synthesized by solid phase phosphoramidite trisaccharide method, 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 the recombinant strain, the recombinant gene can be further amplified on a large scale by culturing the strain. In addition, the recombinant gene may also contain residual sequences of restriction sites, other accessory elements (such as control elements, including promoters, etc.), marker substances (such as fluorescent markers, etc.) and other sequences that do not affect the expression of the target gene.

[0024] "Cloning scar": refers to the introduction of additional sequences in the gene expression region due to the binding of the inserted foreign DNA fragment to the vector during the gene cloning process. 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 therefore cut from the signal peptide by the signal peptidase when passing through the membrane. When cloning constructs after the signal peptide, restriction endonucleases usually require specific sequences to cut the DNA, which leaves a cloning scar after the signal peptide sequence.

[0025] "Signal peptide": A short peptide usually 16-30 amino acids long, present at the N-terminus of most newly synthesized proteins that are destined to enter the secretory pathway. Signal peptides are also called signal sequences, targeting signals, localization signals, localization sequences, transit peptides, leader sequences or leader peptides. Signal peptides are usually cleaved from proteins by signal peptidases.

[0026] "Signal peptide cleavage site": refers to the specific dipeptide position where the 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 an algorithm such as SignalP4.1, which can be used online at http: / / www.cbs.dtu.dk / services / SignalP / (Center for Biological Sequence Analysis, Technical University of Denmark).

[0027] "Promoter" refers to a DNA region located near the start site of gene transcription, usually located upstream of the same chain (pointing to the 5' region of the sense chain), which is responsible for initiating the transcription process of a specific gene and transcribing the gene information into messenger RNA (mRNA). According to their 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 by specific signal pathways or environmental conditions; (2) Constitutive promoters, which are not regulated by any inducer substances and can continuously drive gene transcription regardless of whether the environmental conditions contain inducers. Constitutive promoters maintain basic gene expression levels in cells and are essential for many biological processes.

[0028] Abbreviation "RBS": refers to ribosome-binding site, or the binding site of the ribosome. This is the nucleotide sequence upstream of the start codon of the mRNA transcript that is responsible for recruiting the ribosome during the initiation of protein translation.

[0029] "Expression": refers to the process by which DNA is 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 polynucleic acid fragments in host cells. Many prokaryotic and eukaryotic expression vectors are commercially available, and you can choose a suitable expression vector according to your actual needs.

[0031] "Chassis cell": refers to a host vector used to express the DNA involved in the present invention. The host may include any organism that can contain and express the nucleic acid or gene involved in the present invention, but is not limited thereto. The chassis cell can be a prokaryotic organism or a eukaryotic organism, a single cell or a multicellular cell, including mammalian cells, plant cells, fungi, etc. According to the prior art, those skilled in the art can achieve heterologous expression of the recombinant DNA of the present invention in different disclosed chassis cells by adjusting parameters by a limited number of experiments. The chassis cell can be selected from at least one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansenula, Candida, Rhodotorula, Bacillus, Escherichia, Salmonella, Clostridium, Streptomyces, Staphylococcus, Neisseria, and Shigella. 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 E. coli. Suitable E. coli strains (among many others) 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 E. coli strains are all commercially available strains.

[0032] "Identity" refers to the residues in the two sequences that are the same when aligned for maximum correspondence. For example, if corresponding segments of the two sequences have identical residues at 5 out of 10 positions when properly aligned, the two sequences are said to have 50% identity. Most bioinformatics programs report percent identity for regions of the aligned sequences, which are not usually entire molecules. If the alignment is long enough and contains enough identical residues, an expected value can be calculated, which indicates that the level of identity in the alignment is unlikely to occur by chance.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

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

[0035] In summary, compared with the wild-type laccase (enzyme activity 1.547 U / mg), the enzyme activity of the mutant of the present invention is significantly improved, up to 10 times, which significantly reduces the cost of wastewater treatment. DETAILED DESCRIPTION

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

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

[0038] 1. Oxidation of substrates: Laccase catalyzes the redox reaction of substrates, using molecular oxygen (O2) as an oxidant to transfer electrons from substrates (including starch, phenolic compounds, dyes, etc.) to molecular oxygen, reducing them to water (H2O). Some organic matter in the substrate will be converted into products with low toxicity or biodegradable;

[0039] 2. Participation of copper ions: The catalytic activity of laccase comes from the copper ions it contains. Copper ions play a key role in the active site 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. Produce quinone compounds: For starchy substances, laccase oxidizes them into aldehydes, ketones or quinone compounds. These oxidation products are easier to be further degraded by microorganisms than the original substrates, reducing the pollution load of wastewater;

[0041] 4. Water generation: 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 sequence of the target gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and these sequences were inserted into the expression vector by molecular cloning technology. Specifically, these sequences were cloned into the plasmid pET28a (+) to generate the corresponding recombinant plasmid. Subsequently, these recombinant plasmids were introduced into Escherichia coli BL21 (DE3) cells by transformation, thereby constructing Escherichia coli strains containing different plasmids, i.e., recombinant bacteria. It is worth noting that the prior art can provide a variety of available plasmids and chassis cells, and this embodiment only provides a specific solution.

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

[0046] The recombinant bacteria were inoculated into liquid LB medium (5 g yeast extract, 10 g peptone, 10 g PBS) and cultured at 37 °C in a shaking incubator (speed 220 rpm) until OD 600 The value is about 5.0, and then IPTG is added to a concentration of 200 μM, and the temperature is lowered to 16°C for overnight culture. After overnight culture, the bacteria are collected by centrifugation at 4000 g for 10 min and resuspended in 10% volume of buffer NTA (50 mM Tri-HCl, 300 mM NaCl, pH 8.0). Then, the bacteria are broken by ultrasound, and then centrifuged at 12000 rpm for 1h, and the supernatant is taken as the crude enzyme solution. The prior art can provide a variety of available methods for inducing recombinant bacterial expression, and this embodiment only provides a specific scheme.

[0047] (3) The crude enzyme solution is passed through Ni 2+ The resin is subjected to affinity chromatography. Since the expressed target protein C-terminus carries a histidine tag, it can be specifically bound to the resin. First, the resin is rinsed with buffer NTA and imidazole buffer to remove unbound impurities, and then eluted with buffer NTA, and the eluate containing the target protein is collected. The resulting solution is dialyzed to remove imidazole, and the molecular weight cutoff of the dialysis bag is 1kDa. After dialysis, the solution is concentrated until the protein concentration reaches 0.2 mg / mL, and then freeze-dried to obtain laccase powder. According to the prior art, those skilled in the art can adjust the parameters by a limited number of experiments to achieve the purification of laccase, which is not described in detail here. It is worth noting that a variety of available laccase purification methods are provided in the prior art, and this embodiment only provides a specific scheme.

[0048] According to the above laccase preparation method, wild-type laccase (LAC-1, SEQ ID NO: 1) and laccase mutants (LAC-2 to LAC-7) were heterologously expressed and purified.

[0049] Example 2 Enzyme activity determination:

[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. Determination of different laccase activities:

[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, and ABTS was converted into ABTS free radicals under the catalytic action of laccase. Since the absorption coefficient of ABTS free radicals at 420nm was significantly higher than that of substrate ABTS, the absorbance value gradually increased with the increase of ABTS free radical concentration. Therefore, by detecting the change of OD value at 420nm, the enzyme activity of wild-type laccase or laccase mutant can be determined.

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

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

[0055]

[0056] As shown in Table 1, the enzyme activity of the wild-type laccase is relatively low, only 1.547 U / mg, while the enzyme activity of the laccase mutants obtained by multiple groups of mutations in the present invention is significantly improved, all above 10 U / mg.

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

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

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

[0060]

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

[0062]

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

[0064]

[0065] Table 5 Enzyme activity 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] It can be seen from Tables 2 to 7 that the laccase variants LAC-2 to LAC-7 can maintain high enzyme activity under the conditions of pH 5.0 and 15 to 50°C.

[0072] Example 4 Determination of laccase mutant activity under different pH conditions

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

[0074] Table 8 Enzyme activity of laccase variant LAC-2 at different pH

[0075]

[0076] Table 9 Enzyme activity of laccase variant LAC-3 at different pH

[0077]

[0078] Table 10 Enzyme activity of laccase variant LAC-4 at different pH

[0079]

[0080] Table 11 Enzyme activity of laccase variant LAC-5 at different pH

[0081]

[0082] Table 12 Enzyme activity of laccase variant LAC-6 at different pH

[0083]

[0084] Table 13 Enzyme activity of laccase variant LAC-7 at different pH

[0085]

[0086] It can be seen from Tables 8 to 13 that the laccase variants LAC-2 to LAC-7 can maintain high enzyme activity under the conditions of 25°C and pH 4.0 to 7.0.

[0087] Example 5 Determination of laccase mutant 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 put into the same reaction system. The measurement results are shown in Table 14:

[0089] Table 14 Enzyme activities of laccase mutants under different temperature and pH conditions

[0090]

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

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: 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, wherein the temperature of the desizing wastewater is 15-50° C. and the pH value is 4-7.

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