Cutinase mutant and application thereof in degradation of polyethylene glycol terephthalate

Abstract

The invention discloses a cutinase mutant and application thereof in degradation of polyethylene glycol terephthalate, and belongs to the field of environmental science. According to the present invention, BhrPETase derived from bacteria HR29 and LC cutinase of a metagenome in plant compost are modified to obtain mutants, the mutants are subjected to single-point or multi-point mutation near the substrate binding site of the BhrPETase or the LC cutinase, and the 184th site and the 156th site are subjected to single-point mutation or combined mutation to construct six mutants; compared with a wild type enzyme, the six mutants (M1-M6) have the advantages that the enzyme activity and the PET degradation efficiency are obviously improved, and the industrial prospect is good.

Description

Technical Field

[0001] This invention relates to keratinase mutants and their application in the degradation of polyethylene terephthalate, belonging to the field of enzyme engineering technology. Background Technology

[0002] Polyethylene terephthalate (PET) is a type of plastic that has been widely used worldwide due to its advantages such as high mechanical strength, low permeability, light weight, and low cost. However, the large amount of PET waste that is not effectively recycled and is difficult to degrade naturally leads to its accumulation in the global ecosystem, causing nutrient loss and soil compaction. Furthermore, the large amounts of plastic floating in the ocean pose a devastating threat to marine ecosystems. Currently, landfilling, incineration, and physicochemical recycling methods waste energy and have extremely serious environmental impacts. Therefore, biodegradation is considered the most effective method for controlling plastic pollution.

[0003] Various PET hydrolases have been identified and proven to degrade PET to varying degrees, such as PETase from Ideonella sakaiensis, LC cutinase from metagenomic sources in plant compost, cutinase from Saccharomonoraviridis AHK190, HiC from Thermomyces insolens, and lipase B from Candida antarctica. Although some identified PET hydrolases have shown relatively high degradative activity, their degradation efficiency is far from meeting the requirements for large-scale degradation.

[0004] In 2018, Kato et al. studied BhrPETase, a PET hydrolase derived from bacteria HR29. It exhibits good heat resistance and can catalyze the hydrolysis of ester bonds, effectively degrading PET into terephthalic acid (TPA) and ethylene glycol (EG). However, the enzyme's catalytic efficiency is relatively low. Therefore, site-directed mutagenesis is needed to modify the enzyme's structure to improve its PET degradation efficiency and achieve highly efficient PET biodegradation. Summary of the Invention

[0005] The purpose of this invention is to overcome the low efficiency of keratinase in degrading PET and to provide a new mutant of keratinase and its applications. This invention synthesizes BhrPETase, derived from bacteria HR29, which can hydrolyze PET, and expresses and purifies it. After studying the structure of BhrPETase, mutations were made in the amino acids involved in substrate interactions in its active region to increase the enzyme's activity against the substrate PET. Furthermore, it was demonstrated that this mutation is also applicable to metagenomic LC keratinase in plant compost.

[0006] The present invention provides a keratinase mutant, wherein the mutant has substituted at least one amino acid at positions 184 and 156 of the starting sequence; wherein the starting sequence has at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptides at positions 1 to 259 shown in SEQ ID NO: 1 and SEQ ID NO: 5.

[0007] In one embodiment, the mutant substitutes at least one amino acid in the starting sequence (Bhr keratinase) shown in SEQ ID NO: 1 as follows: Replace histidine at position 184 with asparagine; Replace tryptophan with tyrosine at position 156; In one embodiment, the mutant is a BhrPETase mutant M1(H184N) in which the 184th amino acid of the polypeptide shown in SEQ ID NO: 1 is replaced by asparagine instead of histidine, and its amino acid sequence is shown in SEQ ID NO: 2.

[0008] In one embodiment, the mutant is a BhrPETase mutant M2 (W156Y) in which the 156th amino acid of the polypeptide shown in SEQ ID NO: 1 is mutated from tryptophan to tyrosine, and its amino acid sequence is shown in SEQ ID NO: 3.

[0009] In one embodiment, the mutant is a BhrPETase mutant M3 (H184N / W156Y) in which the 184th amino acid of the polypeptide shown in SEQ ID NO: 1 is mutated from histidine to asparagine and the 156th amino acid is mutated from tryptophan to tyrosine. Its amino acid sequence is shown in SEQ ID NO: 4.

[0010] In one embodiment, the mutant substitutes at least one amino acid in the starting sequence (LC keratinase) shown in SEQ ID NO: 5 as follows: Replace histidine at position 184 with asparagine; Replace tryptophan with tyrosine at position 156; In one embodiment, the mutant is a polypeptide whose 184th amino acid, histidine, is replaced with asparagine and named LC keratinase mutant M4(H184N), the amino acid sequence of which is shown in SEQ ID NO: 6.

[0011] In one embodiment, the mutant is a polypeptide with the 156th amino acid changed from tryptophan to tyrosine, named LC keratinase mutant M5(W156Y), whose amino acid sequence is shown in SEQ ID NO: 7.

[0012] In one embodiment, the mutant is a polypeptide whose 184th amino acid is mutated from histidine to asparagine and whose 156th amino acid is mutated from tryptophan to tyrosine, named LC keratinase mutant M6 (H184N / W156Y), and whose amino acid sequence is shown in SEQ ID NO: 8.

[0013] The present invention provides a polynucleotide encoding the mutant.

[0014] This invention provides nucleic acid constructs and vectors containing the polynucleotides described herein.

[0015] In one embodiment, the carrier includes, but is not limited to, pET series, Du et series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series carriers.

[0016] In one embodiment, the vector is pET 24a, and the polynucleotide encoding the mutant is inserted into the polyclonal restriction site of the pET24a vector.

[0017] The present invention provides a host cell that expresses the mutant or contains the polynucleotide.

[0018] In one embodiment, the host cell includes, but is not limited to, Escherichia coli, Pichia pastoris, Bacillus subtilis, and Saccharomyces cerevisiae.

[0019] The present invention provides a method for producing the keratinase mutant.

[0020] In one embodiment, the method includes: a) Culturing the host cells of the present invention under conditions suitable for expressing the variants; and b) Optionally, the variants will be recycled.

[0021] In one embodiment, a) is to culture the host cells in a culture medium containing a carbon source, a nitrogen source, and inorganic salts.

[0022] In one embodiment, step a) involves culturing the host cells in LB medium at 35-40°C and 150-250 rpm until OD600 = 0.8 ± 0.5, then adding IPTG to a final concentration of 0.1 mM, and culturing at 20-30°C and 150-250 rpm to obtain a fermentation broth.

[0023] In one embodiment, b) involves centrifuging the fermentation broth and collecting the supernatant to obtain a crude enzyme solution, which is then purified by a nickel column to obtain a pure enzyme solution.

[0024] The present invention provides a product containing the aforementioned keratinase mutant.

[0025] In one embodiment, the product includes, but is not limited to, enzyme preparations or compositions containing the keratinase mutant.

[0026] In one embodiment, the enzyme preparation or composition contains an enzyme protectant; the protectant includes an acid-base regulator and a lyophilization protectant.

[0027] The present invention provides a method for degrading polyethylene terephthalate or substances containing polyethylene terephthalate, wherein the method comprises adding the keratinase mutant to a system containing polyethylene terephthalate for reaction.

[0028] In one embodiment, the amount of the keratinase mutant added to the system is not less than 300 U / g substrate; optionally, the amount of the keratinase added is 300~400 U / g substrate.

[0029] In one embodiment, the reaction is carried out at pH 8.0 ± 0.5, 65–75 °C, and 150–250 rpm.

[0030] In one implementation, the reaction time is not less than 3 hours.

[0031] The present invention also provides compositions containing the said keratinase mutant.

[0032] In one embodiment, the composition uses the keratinase mutant as the main enzyme component.

[0033] In one embodiment, the composition may contain multiple enzymatic activities, include the keratinase mutant, and contain one or more components selected from the group consisting of proteases, glucosylamylase, β-amylase, etc. Amylase, amylopectin.

[0034] The present invention also provides the use of the mutant in the degradation of polyethylene terephthalate or in products containing polyethylene terephthalate.

[0035] In one embodiment, the product is a plastic product containing polyethylene terephthalate.

[0036] In one embodiment, the plastic products include, but are not limited to, rigid or flexible packaging, agricultural films, bags, disposable items, textiles, fabrics, nonwovens, floor coverings, plastic waste, or fiber waste.

[0037] In one embodiment, the plastic product is a plastic film, a plastic bottle, or a plastic tray.

[0038] The beneficial effects of this invention are: This invention modifies BhrPETase derived from bacteria HR29 and LC keratinase from plant compost to obtain mutants. The mutants are constructed by single or double point mutations near the substrate binding site of the BhrPETase or LC keratinase, and by single or combined mutations at positions 184 and 156, resulting in six mutants. Compared to wild-type BhrPETase, the three BhrPETase mutants (M1, M2, and M3) described in this invention exhibit significantly improved enzyme activity, PET specific enzyme activity, and PET degradation efficiency, demonstrating promising industrial potential. Attached Figure Description

[0039] Figure 1 This is an SDS-PAGE image of the mutant. Detailed Implementation

[0040] Definitions or terms: Keratinase: The term "cutinase" refers to an enzyme in class EC3.1.1.74 as defined by enzyme nomenclature. For the purposes of this invention, cutinase activity is determined according to the procedure described in the examples. In one aspect, variants of the invention have at least 20%, for example, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the cutinase activity of the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 5.

[0041] Polynucleotides encoding mutants: The term "polynucleotides encoding mutants" directly determines the amino acid sequence of the keratinase variant. The boundaries of the coding sequence are typically defined by a read frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof. The polynucleotides involved in this invention include, but are not limited to: SEQ ID NO: 9 (encoding wild-type BhrPETase), SEQ ID NO: 10 (encoding wild-type LC keratase), SEQ ID NO: 11 (encoding M1(H184N) mutant), SEQ ID NO: 12 (encoding M2(W156Y) mutant), SEQ ID NO: 13 (encoding M3(H184N / W156Y) mutant), SEQ ID NO: 14 (encoding M4(H184N) mutant), SEQ ID NO: 15 (encoding M5(W156Y) mutant), and SEQ ID NO: 16 (encoding M6(H184N / W156Y) mutant).

[0042] Expression: The term “expression” includes any step involved in the production of keratinase variants, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0043] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule containing a polynucleotide encoding a keratinase variant of the present invention and operatively linked to a control sequence provided for its expression.

[0044] Fragment: The term "fragment" means a polypeptide that has one or more (e.g., several) amino acids missing from its amino and / or carboxyl termini; said fragment has keratinase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%, of the number of amino acids 1 to 331 (i.e., excluding the zymogen region sequence length) of SEQ ID NO: 1 or SEQ ID NO: 5.

[0045] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0046] The host cell can be any cell that is useful in the recombinant production of keratinase variants, such as prokaryotic or eukaryotic cells.

[0047] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Bacillus macrocephala, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0048] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0049] Improved degradability: The term “improved degradability” refers to the characteristics of a keratinase variant that is more effective than the parent keratinase in degrading PET or acting on ester bonds.

[0050] Isolated: The term “isolated” means a substance in a form or environment that does not exist in nature. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance removed at least partially from one or more naturally occurring components associated with it in nature, including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to such a substance found in nature; or (4) any substance modified by increasing the amount of said substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding said substance; use of a promoter stronger than the promoter naturally associated with the gene encoding said substance). Isolated substances may be present in fermentation broth samples.

[0051] Variant: The term "variant" refers to a polypeptide having keratinase activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. The variants of the present invention have the polypeptide amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 4, with substitutions occurring at positions 184 and 156, respectively, where the substituted amino acids are asparagine (N) and tyrosine (Y). The variants of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the keratinase activity.

[0052] M1(H184N): Based on the wild type shown in SEQ ID NO: 1, the 184th position is replaced with asparagine (N).

[0053] M2(W155Y): Based on the wild type shown in SEQ ID NO: 1, position 156 is replaced with tyrosine (Y).

[0054] M3(H184N / W155Y): Based on the wild type shown in SEQ ID NO: 1, positions 184 and 156 are replaced with asparagine (N) and tyrosine (Y), respectively.

[0055] M4(H184N): Based on the wild type shown in SEQ ID NO: 5, the 184th position is replaced with asparagine (N).

[0056] M5(W155Y): Based on the wild type shown in SEQ ID NO: 5, position 156 is replaced with tyrosine (Y).

[0057] M6(H184N / W155Y): Based on the wild type shown in SEQ ID NO: 5, positions 184 and 156 are replaced with asparagine (N) and tyrosine (Y), respectively.

[0058] In the variations describing the invention, the nomenclature described below has been adapted for ease of reference, using the accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0059] Substitution: For amino acid substitution, use the following nomenclature: original amino acid, position, substituted amino acid.

[0060] The keratinase of the present invention comprises a substitution at position 184 corresponding to the sequence shown in SEQ ID NO: 1; SEQ ID NO: 5, substituted to form asparagine (N); and a substitution at position 156, substituted to form tyrosine (Y); wherein, i) The keratinase described herein has at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptides at positions 1 to 259 shown in SEQ ID NO: 1 and SEQ ID NO: 5; and / or ii) The keratinase is a polypeptide encoded by a polynucleotide having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the mature polypeptide coding sequences of nucleotides 1 to 777 shown in SEQ ID NO: 9 and SEQ ID NO: 10. In one embodiment, the keratinase, at position 184, is substituted to form asparagine (N); In one embodiment, the keratinase has a substitution at position 156, which is replaced with tyrosine (Y). Fermentation broth: The term "fermentation broth" refers to a preparation produced by cell fermentation that undergoes little or no recovery and / or purification. For example, fermentation broth is produced when a microbial culture is incubated to saturation under carbon-limited conditions that allow protein synthesis (e.g., enzyme expression by the host cell) and secretion of proteins into the cell culture medium. The fermentation broth may contain unfractionated or fractionated contents of the fermentation material obtained at the end of fermentation. Typically, the fermentation broth is unfractionated and contains used culture medium and cell debris remaining after, for example, removal of microbial cells (e.g., filamentous fungal cells) by centrifugation. In some embodiments, the fermentation broth contains used cell culture medium, extracellular enzymes, and viable and / or non-viable microbial cells.

[0061] Plastics: The term "plastics" or "plastic materials" refers to plastic products (e.g., sheets, trays, films, tubes, blocks, fibers, fabrics, etc.) and plastic compositions used in the manufacture of plastic products. In addition to polymers, plastic materials may also contain other substances or additives, such as plasticizers, mineral or organic fillers, dyes, etc. Therefore, in the context of this invention, plastic materials refer to any plastic product and / or plastic composition containing at least one semi-crystalline and / or amorphous form of polymer, particularly at least one PET.

[0062] Plastic products: The term “plastic products” includes manufactured products containing plastic, such as rigid or flexible packaging (films, bottles, pallets), agricultural films, bags, disposable items, textiles, fabrics, nonwovens, floor coverings, plastic waste or fiber waste, etc.

[0063] Polymer: The term "polymer" refers to a compound whose structure consists of multiple repeating units (i.e., "monomers") linked by chemical covalent bonds. In the context of this invention, unless otherwise specified, "polymer" more specifically refers to such compounds used in compositions of plastic materials.

[0064] Polyester: The term "polyester" refers to a polymer whose main chain contains ester functional groups. Ester functional groups are characterized by bonds between carbon and three other atoms: a single bond with another carbon atom, a double bond with oxygen, and a single bond with another oxygen atom. The oxygen atom, bonded to carbon via a single bond, is itself bonded to another carbon via a single bond. Polyesters can be made from only one type of monomer (i.e., homopolymer) or at least two different monomers (i.e., copolymer). Polyesters can be aromatic, aliphatic, or semi-aromatic. For example, polyethylene terephthalate (PET) is a semi-aromatic copolymer composed of two monomers, terephthalic acid and ethylene glycol.

[0065] Degradation: "Degradation" of plastics or PET-containing plastics refers to the degradation of the polymer in a plastic material into smaller molecules, such as monomers and / or oligomers. In the context of this invention, degradation of PET or PET-containing plastic materials refers to the degradation of PET in the plastic into monomers (such as terephthalic acid and / or ethylene glycol) and / or oligomers; said oligomers include, but are not limited to, dimethyl terephthalate (DMT), terephthalic acid 2... Hydroxyethyl methyl ester (MHET), bis(2-hydroxyethyl) terephthalate Hydroxyethyl ester (BHET).

[0066] Materials involved in the following embodiments: The polyethylene terephthalate (PET) plastic used in the following examples was purchased from Goodfellow. BHET, MHET, and TPA were purchased from Sigma. The amorphous PET powder was an amorphous film purchased from Gold Fellow, which was powdered using liquid nitrogen.

[0067] The *E. coli* JM109 and *E. coli* BL 21(DE3) mentioned in the following examples were purchased from Takara. Baori Biotechnology (Beijing) Co., Ltd.

[0068] The culture media involved are as follows: LB solid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, agar 13, pH 7.0.

[0069] LB liquid medium (g / L): peptone 10, yeast extract 5, sodium chloride 10, pH 7.0.

[0070] The detection methods involved in the following embodiments are as follows: Protein concentration determination method: Protein concentration was determined using the Coomassie Brilliant Blue method (Analytical Biochemistry 1976 72(1)). 2): 248 54).

[0071] Methods for detecting degradation products and their content: Standard preparation: Weigh out the TPA, MHET and BHET standards respectively and dissolve them in dimethyl sulfoxide (DMSO) to prepare a stock solution. Dilute the stock solution with sterile water to a standard solution of 0.1 mg / mL. Filter the solution through a 0.22 μM filter and inject it into the liquid chromatography bottle for HPLC detection. Sample preparation: Let the culture medium stand for 10 min, take 5 mL of supernatant, centrifuge at 12000 rpm for 8 min, filter with a 0.22 μM filter, inject into the HPLC bottle with a syringe, and perform HPLC detection.

[0072] Degradation rate detection method: Degradation rate (%) = ((m1 / x1 + m2 / x2 + m3 / x3) × M) / (mass of PET before treatment) × 100; m1, m2, and m3 represent the weights of TPA, MHET, and BHET in the reaction mixture volume; M is the relative molecular mass of the PET unit; x1 is the relative molecular mass of TPA; x2 is the relative molecular mass of MHET; and x3 is the relative molecular mass of BHET.

[0073] Methods for detecting enzyme activity: Tri s HCl buffer (10 mM pH 7.0): Accurately weigh 1.2 10 g Tris and 0.5 84 g NaCl, add to about 800 mL of deionized water, stir thoroughly to dissolve, adjust the pH to 8.0 with HCl, and bring the volume to 1000 mL.

[0074] Substrate (50 mmol / L p-nitrophenylbutyrate solution): Accurately weigh 0.1046 g of p-nitrophenylbutyrate and dilute to 10 mL with acetonitrile. Store at 20℃.

[0075] Accurately transfer 1.5 ml of Tris using a 5 mL pipette. Pour HCl buffer (pre-incubated at 37°C for 10 min) into a 0.5 cm glass cuvette and zero the instrument at an absorption wavelength of 405 nm. Take 1.44 mL of Tris... Pour HCl buffer into a 0.5 cm quartz cuvette. Add 30 μL of the diluted enzyme solution to the cuvette, followed by 30 μL of the substrate solution. Mix well and immediately place the cuvette in a visible spectrophotometer. Measure the absorbance (A) at 405 nm. Record the A value every 5 seconds for 1 minute. Calculate the enzyme activity based on the absorbance.

[0076] Enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of p-nitrophenylbutyrate to produce 1 μmol of p-nitrophenol per minute at 65°C and pH 8.0. One enzyme activity unit (U) is defined as the amount of enzyme that catalyzes the hydrolysis of p-nitrophenylbutyrate to produce 1 μmol of p-nitrophenol per minute.

[0077] Methods for producing keratinase mutants: A method for producing a keratinase variant includes: (a) culturing host cells of the present invention under conditions suitable for expressing the keratinase variant; and (b) recovering the keratinase variant.

[0078] Host cells are cultured in a nutrient medium suitable for producing keratinase variants using methods known in the art. For example, cells can be cultured by shake-flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the keratinase or variant. Using procedures known in the art, the culture occurs in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to a disclosed composition. If the keratinase variant is secreted into the nutrient medium, the keratinase variant can be recovered directly from the medium. If the keratinase variant is not secreted, it can be recovered from cell lysates.

[0079] Keratinase variants can be detected using methods known in the art that are specific to keratinase variants. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of keratinase variants (as described in the examples).

[0080] Keratinase variants can be recovered using methods known in the art. For example, keratinase variants can be recovered from nutrient media through routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0081] Keratinase variants can be purified to obtain substantially pure keratinase variants using a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), and SDS-PAGE. PAGE or extraction.

[0082] Alternatively, instead of recycling the keratinase variant, the host cells of the present invention expressing the keratinase variant are used as the source of the keratinase variant.

[0083] Example 1: Construction of mutant recombinant plasmid (1) Mutations were performed on wild-type Bhr keratinase (amino acid sequence as shown in SEQ ID NO: 1) and wild-type LC keratinase (amino acid sequence as shown in SEQ ID NO: 5), respectively. The nucleotide sequences shown in SEQ ID NO: 9 (encoding Bhr keratinase) and SEQ ID NO: 10 (encoding LC keratinase) were inserted into pET 24a using restriction endonucleases. Nde I site and Xho Site I yielded pET 24a BhrPETase and pET 24a LC keratinase; then using site-directed mutagenesis technology (site directed mutagenesis), with pET24a BhrPETase and pET 24a Using LC keratinase plasmid as a template, PCR was performed using the primers shown in Table 1 to obtain the target gene mutation fragment (primers are shown in Table 1). Megawhop PCR was then used to combine the target fragment with pET... The 24a expression vector was ligated to obtain a recombinant plasmid. The constructed recombinant plasmid was then transformed into *E. coli* (…). Escherichia coli JM1 09 was used to obtain the transformation product; the transformation product was spread on LB solid medium (containing 40 μg / mL kanamycin) and incubated upside down in a 37℃ incubator for 8-12 h to obtain transformants; the transformants were picked and inoculated into LB liquid medium and cultured in shake flasks at 37℃ and 120-180 rpm for 8-12 h, and then the plasmids were extracted for sequencing verification to obtain recombinant plasmids expressing different mutants.

[0084] Table 1 Primer sequences

[0085] (2) Construction of mutant recombinant Escherichia coli Transform the correct recombinant plasmid obtained in step (1) into Escherichia coli ( Escherichia coli BL21 was used to obtain the transformation product. The transformation product was spread on LB solid medium (containing 50 μg / mL kanamycin) and incubated upside down in a 37℃ incubator for 8–12 h to obtain transformants. Transformants were picked and inoculated into LB liquid medium and cultured in shake flasks at 37℃ and 120–180 rpm for 8–12 h. Plasmids were then extracted for enzyme digestion and sequencing verification. Correct verification yielded recombinant *E. coli* BL21 / pET. 24(+) M1, E. coli BL21 / pET 24(+) M2, E. coli BL21 / pET 24(+) M3, E. coli BL2 1 / pET 24(+) M4, E. coli BL2 1 / pET 24(+) M5, E. coli BL2 1 / pET 24(+) M6.

[0086] (3) Preparation of mutants The mutant recombinant *E. coli* obtained in step 2 was transferred to 100 mL of LB liquid medium at an inoculation rate of 5% (v / v). The medium was incubated at 37°C and 200 rpm for 3 hours until OD600 = 0.8. IPTG was then added to a final concentration of 0.1 mM, and the medium was further incubated at 25°C and 200 rpm for 20 hours to obtain the fermentation broth. The broth was centrifuged at 8000 rpm for 15 minutes to obtain the supernatant, which is the crude enzyme solution. Figure 1 As shown, the crude enzyme solution contains the target protein. To quickly obtain high-purity enzyme protein, the above crude enzyme solution was first precipitated with ammonium sulfate to obtain protein precipitate, and then precipitated with 10mM pH 7.0 Tris... The enzyme solution was reconstituted with HCl buffer and then eluted using a nickel column to obtain the purified enzyme solution.

[0087] The recombinant plasmid pET 24a BhrPETase and pET 24a LC keratinase was used to transform Escherichia coli according to step (2) to obtain wild-type recombinant Escherichia coli; mutants were prepared according to step (3) to obtain wild-type crude enzyme solution and purified enzyme solution.

[0088] Example 2: Expression of the mutant Enzyme activity was determined in crude enzyme solutions of wild-type and mutant strains using a continuous spectrophotometric method to assess the hydrolytic activity of PETase and its mutant pNPB esterases. The total reaction volume was 1.5 mL, containing 100 mM phosphate buffer (pH 8.0) (1.44 mL), the enzyme solution to be tested (30 μL), and pNPB at a final concentration of 50 mM (30 μL). The amount of p-nitrophenol produced over 60 s was recorded at 405 nm. One unit (U) of enzyme activity was defined as the amount of enzyme required to hydrolyze the pNPB substrate and produce 1 μM p-nitrophenol product over 60 s at 60 °C and pH 8.0.

[0089] As shown in Table 2, compared with the wild type, the activities of the four mutants were significantly improved. For example, the enzyme activity of mutant M2 increased to 52.3 U / mL, an increase of 63.4%, compared with 32.0 U / mL of the wild type. At the same time, the expression levels of the mutants were also increased to varying degrees. Compared with 0.08 mg / mL of the wild type, the protein concentration of mutant M2 increased to 0.12 mg / mL, an increase of 50%.

[0090] Table 2 Enzyme activity and protein concentration of BhrPETase mutants

[0091] Example 3: Degradation performance of mutants on PET The purified enzyme solution obtained in Example 1 was collected, and the specific activity of the pure enzyme to PET and its degradation performance on PET were tested.

[0092] Determination of PET specific activity: The reaction system consisted of 100 mg amorphous PET powder, 0.1 mg pure enzyme, 1 mL of 1M pH 8.0 phosphate buffer, and deionized water to a final volume of 10 mL. During the reaction, 210 mg CaCO3 was added to maintain the pH at 7.5–8.0. The reaction was carried out at 60 °C and 200 rpm in a water bath for 10 min. An untreated sample was used as a control. Methanol was added to terminate the reaction before sampling. The supernatant was collected after centrifugation at 12000 rpm for 5 min, filtered through a 0.22 μm filter, and finally analyzed by high-performance liquid chromatography (HPLC). Each independent experiment was performed in triplicate. Substrate consumption was ensured to be no more than 10%. Enzyme activity was calculated by detecting PTA content; one unit (U) of enzyme activity was defined as the production of 1 μM PTA within 1 min at 60 °C.

[0093] Determination of PET degradation rate: Take 10 mL of pH 8.0, 0.1 M phosphate buffer, add 100 mg of amorphous PET powder to the buffer, and add the mutant obtained in Example 2 at an addition rate of 1 mg enzyme protein / g substrate; react in a constant temperature water bath shaker at 60℃ and 200 rpm for 3 h, boil the reaction solution for 15 min to inactivate the enzyme, centrifuge at 12,000 rpm / min for 10 min to obtain the supernatant for HPLC analysis to calculate the degradation rate.

[0094] The results are shown in Table 3. The four mutants showed significantly higher PET degradation efficiency than the wild-type protein. Among them, the BhrPETase mutant M2 showed a 29.43% higher degradation rate of PET substrate over 3 hours compared to the wild-type.

[0095] Table 3. Effects of BhrPETase mutants on PET degradation

[0096] Example 4: Compositions or enzyme preparations containing mutants Keratinase protein was prepared according to the method in Example 1, and the enzyme protein was mixed with an enzyme protectant; the protectant included an acid-base regulator and a lyophilization protectant.

[0097] The pH adjuster is a conventional buffer solution that can maintain pH, such as malate-sodium malate buffer, acetate-sodium acetate buffer, citrate-sodium citrate buffer, citrate-sodium hydroxide-hydrochloric acid buffer, and Tris-hydrochloric acid buffer. The buffer solution is used to maintain the keratinase protein at its optimal pH conditions, thereby ensuring its maximum catalytic activity.

[0098] The freeze-drying protectant is used to reduce the chemical and / or physical instability of keratinase proteins during freeze-drying and / or subsequent storage, including but not limited to sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, aritol, xylitol, sorbitol, mannitol; amino acids, such as arginine or histidine; soluble salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol) or poly(propylene glycol); and combinations of any of the above.

[0099] Example 5: Application of keratinase mutants The keratinase mutant involved in this invention can be used to degrade and recycle polyesters, such as polyethylene terephthalate (PET), by breaking its ester bonds, thereby degrading the substance polymerized from polyethylene terephthalate and changing it from an insoluble state to a soluble state.

[0100] The keratinase mutant can be used to treat PET-containing textiles. The treated polyester textiles can increase wearing comfort, increase water permeability, reduce static electricity, and improve hand feel and softness.

[0101] The keratinase mutant can be used to improve the functional coating of PET-containing yarns or fabrics. Treating yarns or fabrics with the keratinase mutant of the present invention can increase the number of functional groups on the surface of the fabric, thereby adsorbing functional coating agents.

[0102] The keratinase mutant can also be used for lipases and other known applications of keratinase, such as in the paper industry (patent publication CN1 0648077 1A), the leather, wool and related industries (patent publication CN1 04673 772A), and for other applications related to defatting / removal. The immobilized keratinase mutant can be used as a catalyst in organic synthesis (such as esterification, transesterification, or lipid hydrolysis) in the lipid and oil manufacturing industries.

[0103] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A keratinase mutant, characterized in that, Based on the keratinase starting sequence, mutations are made at position 184 and / or position 156; the keratinase starting sequence is shown in SEQ ID NO: 1 or SEQ ID NO:

5.

2. The keratinase mutant according to claim 1, characterized in that, It has any of the mutations (a) to (f); (a) Mutate histidine at position 184 of the amino acid sequence shown in SEQ ID NO: 1 to asparagine; (b) Mutate tryptophan at position 156 of the amino acid sequence shown in SEQ ID NO: 1 to tyrosine; (c) Mutate histidine at position 184 of the amino acid sequence shown in SEQ ID NO: 1 to asparagine and mutate tryptophan at position 156 to tyrosine; (d) Mutate histidine at position 184 of the amino acid sequence shown in SEQ ID NO: 5 to asparagine; (e) Mutate tryptophan at position 156 of the amino acid sequence shown in SEQ ID NO: 5 to tyrosine; (f) Mutate histidine at position 184 of the amino acid sequence shown in SEQ ID NO: 5 to asparagine and mutate tryptophan at position 156 to tyrosine.

3. A polynucleotide encoding the mutant of claim 1 or 2.

4. A nucleic acid construct or vector carrying the polynucleotide of claim 3.

5. A host cell expressing the mutant of claim 1 or 2, or carrying the polynucleotide of claim 3.

6. A recombinant Escherichia coli, characterized in that, Using pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series plasmids as vectors, the mutants described in claim 1 or 2 are recombinantly expressed.

7. A method for degrading polyethylene terephthalate or substances containing polyethylene terephthalate, characterized in that, The mutant of claim 1 or 2, or the fermentation broth of the recombinant Escherichia coli of claim 6, is added to a system containing polyethylene terephthalate for reaction.

8. The method according to claim 7, characterized in that, The substances containing polyethylene terephthalate include rigid or flexible packaging, agricultural films / bags, textiles, fabrics, nonwovens, floor coverings, plastic waste, or fiber waste.

9. The method according to claim 7, characterized in that, The substance containing polyethylene terephthalate includes plastic products; the plastic products include plastic films, plastic bottles, disposable lunch boxes or plastic trays.

10. The method according to claim 7, characterized in that, In the system, the amount of the mutant added is not less than 300 U / g substrate.

Images