Cutinase capable of degrading polyurethane plastic as well as extracellular expression method and application of cutinase

Abstract

The invention discloses cutinase ChCut1 with a function of degrading polyurethane plastics. The amino acid sequence of the cutinase is shown as SEQ ID NO. 1. The invention also provides an extracellular expression method and application of the cutinase. The cutinase can be massively expressed in the saccharomyces cerevisiae, so that the efficient, stable and low-cost cutinase is produced. The cutinase can effectively degrade Impranil PU (polyurethane) under the condition of 28 DEG C, so that a plastic polymer structure is destroyed, and biodegradation under a mild condition is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a keratinase that degrades polyurethane plastics, its extracellular expression method, and its application. Background Technology

[0002] Plastics are widely used in various fields of production and daily life due to their excellent performance and low cost. However, traditional plastics (especially polyolefins and polyesters) are difficult to degrade in the natural environment, causing serious "white pollution" and posing a huge threat to soil, water bodies, marine ecosystems, and even human health. Traditional polyurethane plastics have complex chemical structures (including urethane bonds, ester / ether bonds, urea bonds, etc.), high cross-linking properties, and hydrophobicity, making them extremely difficult to biodegrade in the natural environment, and thus a particularly stubborn type of plastic pollution. Traditional physical (landfill, incineration) and chemical (pyrolysis, solvation) treatment methods have disadvantages such as low efficiency, high energy consumption, and high risk of secondary pollution.

[0003] Utilizing microorganisms or their secreted enzymes to degrade plastics (biodegradation) is considered an environmentally friendly and sustainable solution. Enzymes can efficiently and specifically catalyze the breaking of plastic polymer chains under mild conditions (normal temperature and pressure, neutral pH). Cutinase is a class of serine hydrolases, initially discovered in plant pathogenic fungi, used to decompose the cuticle of plant epidermis (the main component of which is cutin, a polyester). Studies have shown that cutinase has a broad substrate spectrum, capable of hydrolyzing not only natural polyesters (cutin, natural waxes) but also various synthetic polyesters, including polyethylene terephthalate (PET), polylactic acid (PLA), and polyurethane (PU). Its mechanism of action mainly involves hydrolyzing ester bonds (for polyester-type PU) and potential urethane bonds (lower efficiency, but a possibility) in the polyurethane molecular chain, thereby disrupting the polymer structure and achieving depolymerization.

[0004] While keratinases have shown potential in degrading polyurethane (PU), their large-scale production and application costs are key bottlenecks restricting their practical application. Currently, recombinant keratinases are mainly expressed intracellularly in microbial cells (such as bacteria and yeast). Intracellular expression faces challenges such as complex and expensive downstream processing, enzyme activity loss, and limited production efficiency.

[0005] Therefore, developing an efficient, stable, and low-cost method for the extracellular expression of this keratinase in microorganisms, and providing its application, is of great scientific significance and practical value for overcoming the cost bottleneck of its large-scale application in the field of polyurethane plastic biodegradation. Summary of the Invention

[0006] The purpose of this invention is to provide a keratinase capable of degrading polyurethane plastics, and another purpose is to provide an extracellular expression method and application of this keratinase. This allows for the large-scale expression of the keratinase in *Saccharomyces cerevisiae*, enabling the production of a highly efficient, stable, and low-cost keratinase. This invention is achieved through the following technical solutions:

[0007] To achieve the above objectives, the specific plan is as follows:

[0008] The keratinase provided by this invention is derived from Cladosporium halotolerans 6UPA1, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2021967.

[0009] This invention assembled the genome of Cladosporium halotolerans6UPA1 using genome annotation and annotated its gene structure using AUGUSTUS software. By comparing with the CAZY database, a gene encoding a potential keratinase function was successfully identified.

[0010] A keratinase, ChCut1, which degrades polyurethane plastics, has the amino acid sequence shown in SEQ ID NO. 1.

[0011] MKFTAAAIAVLASTAAALPTAVERRQFGTRVGSTVNELTNGACRPITFIFARGS

[0012] TEIGNIGSTVGPPTCEGLKSEYGANQVACQGVGSPYEATIGANALPEGTTSA

[0013] AYGEAQRLFNLASTKCPDTIIVAGGYSQGAAVMTAAVRRLSSSVQDKVAGV

[0014] VLYGNTRNAQNNGKIPNFPPEKALTFCNLTDGVCGGGLVVTAGHLTYTRDVDDAVDYLNERITAAGGI,

[0015] It encodes 224 amino acids and has a theoretical molecular weight of 23.0 kDa.

[0016] A gene encoding a keratinase ChCut1 that degrades polyurethane plastics, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0017] The gene is 672 bp in length and contains 64% G+C.

[0018] The extracellular expression method and application of keratinase provided by this invention comprises the following steps:

[0019] (a) Provides an extracellular signal peptide sequence with the following amino acid sequence: SEQ ID NO.3, MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVL PFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA, encoding 89 amino acids with a theoretical molecular weight of 9.3 kDa;

[0020] (b) The complete keratinase gene ChCut1 (SEQ ID NO:2) from Cladosporium halotolerans 6UPA1 and the above signal peptide were cloned into the expression vector pGADHL, retaining their natural structural domains, and a recombinant plasmid was constructed.

[0021] (c) Using yeast as a host, recombinant plasmids were introduced via lithium acetate conversion.

[0022] (d) Transformants were screened and induced to express using SD-Leu auxotrophic medium;

[0023] (e) Obtaining cutinase from crude enzyme solution of yeast fermentation broth;

[0024] (f) The crude enzyme solution is mixed with Impranil PU and then incubated at 28°C for 24-72 hours. The degradation activity is verified by the clarification of the culture medium or the formation of a clear zone.

[0025] Furthermore, the supernatant contains keratinase.

[0026] Furthermore, the yeast strain is *Saccharomyces cerevisiae*.

[0027] The present invention also provides the application of the above-mentioned keratinase ChCut1 in one or two of the following 1) to 2);

[0028] 1) Degradation of polyurethane waste;

[0029] 2) Industrial cleaning and material surface treatment.

[0030] Furthermore, the industrial cleaning and material surface treatment includes cleaning industrial equipment containing polyurethane residues, removing stubborn polyurethane coatings / adhesive stains from the surfaces of instruments or tools, or pretreatment of surfaces for polyurethane impurities on specific materials.

[0031] The above technical solution has the following beneficial effects:

[0032] This invention utilizes the extracellular keratinase ChCut1 heterologously expressed in *Saccharomyces cerevisiae*, which can effectively degrade waterborne polyurethane (Impranil PU) at 28°C, thereby disrupting the polymer structure and achieving biodegradation under mild conditions. Compared to traditional physicochemical treatment methods, this method has advantages such as mild reaction conditions (room temperature, no need for strong acids or alkalis or high temperatures), environmental friendliness, and low cost, providing a feasible solution for the biodegradation of polyurethane waste. Attached Figure Description

[0033] Figure 1 The image shows a PCR electrophoresis diagram of the keratinase ChCut1 gene (M: DL2,000 DNA Marker; 1-3: ChCut1 amplification products).

[0034] Figure 2 The image shows yeast in a liquid yeast culture medium containing Impranil PU. The left side is the blank control, and the right side is the crude enzyme solution of cutinase ChCut1 degrading Impranil PU. The results show that the yeast containing cutinase can grow normally and degrade aqueous Impranil PU, making the culture medium clear.

[0035] Figure 3 The image shows extracellular enzymes incubated in an inorganic salt medium containing Impranil PU. The crude enzyme solution was incubated in an inorganic salt medium containing Impranil PU for enzyme activity detection. The top row shows the wild-type blank control, and the bottom row shows the degradation of Impranil PU by the crude enzyme solution of ChCut1. The results show that the crude enzyme solution can produce a clear zone.

[0036] Figure 4 The image shows the infrared spectra of the depolymerization products of Impranil PU by extracellular keratinase. A comparison of the spectra between the experimental and control groups reveals significant chemical structural changes in the Impranil PU substrate. At 1700 cm⁻¹... -1 The peaks around the left and right were observed to show a significant decrease in absorption. This absorption peak is usually associated with the bending and stretching of the C=O group, and its decrease indicates that the ester bond has been degraded by the keratinase. The breaking of the ester bond further demonstrates the specific degradation ability of the keratinase for the cross-linked structure of polyurethane. The keratinase ChCut1 has the function of biodegrading aqueous Impranil PU. Detailed Implementation

[0037] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0038] Example 1

[0039] PCR amplification of the gene encoding the keratinase ChCut1

[0040] Based on the genomic information of *Cladosporium halotolerans* 6UPA1 and combined with ORF prediction, the full-length sequence of the keratinase gene was obtained. The gene is 672 bp in length, with a G+C content of 64%, and its sequence is SEQ ID NO.1, encoding 224 amino acids, the amino acid sequence of which is SEQ ID NO.2. The full sequence was synthesized by Nanjing Qingke Biotechnology Co., Ltd., ligated into the pET28a plasmid, and transformed into *E. coli* DH5α.

[0041] The primers used to obtain the keratinase target gene were ChCut1-F and ChCut1-R.

[0042] ChCut1-F:

[0043] 5-ACAGCAAATGGGTCGCGGATCCATGAAGTTCACCGCTGCC-3;

[0044] ChCut1-R:

[0045] 5-GGTGGTGGTGGTGGGTGCTCGAGTCAGATGCCACCAGCAGCAGT-3.

[0046] PCR amplification was performed using the high-fidelity enzyme 2×Phanta Max Master Mix (Novizan Biotechnology). The reaction system consisted of 150 μL of 75 μL Master Mix, 7.5 μL each of forward and reverse primers, and 3 μL of plasmid DNA template.

[0047] Reaction conditions: 95℃ pre-denaturation for 120 seconds; 39 cycles (95℃ 10 seconds → 55℃ 30 seconds → 72℃ 90 seconds); final extension at 72℃ for 300 seconds. The PCR product was verified to be of the correct size (approximately 670 bp) by agarose gel electrophoresis, and the target fragment was recovered for later use (Omega gel recovery kit). PCR amplification results are shown below. Figure 1 .

[0048] Construction of recombinant plasmid pGADHL-Cut

[0049] The purified ChCut1 gene fragment and extracellular signal peptide were ligated to the vector pGADHL-Myc (containing ampicillin resistance and Myc tag).

[0050] The pGADHL vector was linearized by double digestion with restriction endonucleases BamHI and XhoI. The digestion system consisted of 50 μL of 10×Cutsmart Buffer, 1 μL of BamHI, 1 μL of XhoI, and 5 μg of pGADHL vector. The reaction was incubated at 37°C for 1 hour. After digestion, the target band was separated by agarose gel electrophoresis and the gel was recovered to obtain the linearized pGADHL vector.

[0051] The target fragment was inserted into the linearized pGADHL vector using a one-step ligation strategy. The ligation system consisted of 10 μL of ligation system containing 4 μL of the target fragment, 3 μL of recovered linearized pGADHL vector, 2 μL of 5×CEII Buffer, and 1 μL of Exnase II. The ligation reaction was carried out at 25 °C for 30 min. The ligation product was then transformed into DH5α competent cells of Escherichia coli.

[0052] The transformed bacterial culture was spread on LB agar plates containing ampicillin and cultured. Single colonies were picked and verified by PCR using primers W303-α-ChCut1-F and W303-α-ChCut1-R. Positive clones containing the correct inserted fragment, namely recombinant plasmid pGADHL-ChCut1 (ChCut1), were successfully obtained through colony PCR screening.

[0053] The primers used to verify that the recombinant plasmid pGADHL-Cut contains the keratinase gene ChCut1 were W303-α-ChCut1-F and W303-α-ChCut1-R.

[0054] W303-α-ChCut1-F:

[0055] 5-CCAAGCTTTGCAAAGGAATTATGAGATTTCCTTCAATTTTTA-3;

[0056] W303-α-ChCut1-R:

[0057] 5-CGATTCATCTGCAGCTCGAGTCAATGATGATGATGATGAT-3.

[0058] Heterologous expression of the keratinase ChCut1 gene in Saccharomyces cerevisiae

[0059] This invention utilizes *Saccharomyces cerevisiae* as a heterologous expression host. A recombinant plasmid pGADHL-ChCut1 (expressing the keratinase gene), containing the keratinase gene from marine cladocerans, is introduced into host cells. This vector contains a leucine selection marker and a Myc tag.

[0060] Plasmid transformation was performed using the lithium acetate transformation method. The specific system consisted of 100 μL of competent Saccharomyces cerevisiae cells, 5 μL of salmon sperm DNA (vector), and 20 μg of recombinant plasmid DNA. After transformation, the cells were plated on SD-Leu leucine-deficient solid medium and cultured at 28°C until transformants grew.

[0061] Positive transformants were cultured in SD-Leu liquid medium at 28°C with shaking for 60 h (280 rpm). Cells were collected by centrifugation and total protein was extracted using a cell disruptor. Western blotting was used to detect the expression product: the primary antibody was Myc-tag (3A10) mAb, and the secondary antibody was Goat Anti-Mouse IgG-HRP. The results showed a specific band at 17-25 kDa (no band was observed in wild-type W303), the size of which matched the theoretical size of the keratinase ChCut1, confirming successful keratinase expression.

[0062] Functional identification of extracellular keratinase

[0063] Option 1 is as follows:

[0064] Prepare YPB liquid culture medium containing 1% Impranil PU (formulation: yeast extract 10 g / L, peptone 20 g / L, Impranil PU 10 g / L, distilled water to 1 L, sterilize at 121℃ for 20 minutes, cool to 50℃, add Impranil PU), and dispense into 1.5 mL EP tubes.

[0065] Inoculation with Saccharomyces cerevisiae containing keratinase and wild-type yeast (initial OD) 600 =0.1), place in a shaker at 28℃ and shake at 120 rpm for 5-7 days. Observe the state of the culture medium and record: changes in color and turbidity.

[0066] See results Figure 2 Transformed group: The color began to fade on day 3, and by day 5 it had completely turned into a pale yellow, clear solution. Wild-type group: Remained milky white and turbid until day 7.

[0067] Option 2 is as follows:

[0068] Saccharomyces cerevisiae containing keratinase and wild-type yeast were cultured separately in YPB liquid medium at 28°C with shaking for 72 h. After centrifugation at 10,000 rpm for 15 min at 4°C, the supernatant was collected as crude enzyme solution (no purification required).

[0069] Preparation of inorganic salt culture medium assay plates containing 1% Impranil PU

[0070] The extracellular crude enzyme solution was added dropwise to the surface of the plate (10 μL per well) and incubated at 37°C for 24 h.

[0071] See results Figure 3 The enzyme solution droplet produced a diffuse transparent zone at which the diameter was positively correlated with the enzyme activity, directly confirming the degradation function of extracellular secreted keratinase.

[0072] Example 2

[0073] Infrared spectral analysis of the hydrolytic ability of extracellular keratinase ChCut1 on polyurethane plastics

[0074] Saccharomyces cerevisiae containing keratinase and wild-type yeast were inoculated into YPB liquid medium containing 1% Impranil PU and cultured at 28°C with shaking for 72 h. The fermentation broth of Impranil PU treated with keratinase ChCut1 was collected for FTIR analysis of degradation products.

[0075] See results Figure 4 The experimental group was at 1700cm -1 The absorption intensity at the peak (characteristic peak of ester bond C=O stretching vibration) was significantly reduced, while the peak remained stable in the control group. Other characteristic peaks did not show significant changes.

[0076] The extracellular keratinase ChCut1 degrades polyurethane molecules by specifically breaking the ester bond (-COO-) in the polyurethane molecule.

[0077] In summary, this invention utilizes the extracellular keratinase ChCut1 heterologously expressed in *Saccharomyces cerevisiae* to effectively degrade waterborne polyurethane (Impranil PU) at 28°C, thereby disrupting the polymer structure and achieving biodegradation under mild conditions. Compared to traditional physicochemical treatment methods, this method offers advantages such as mild reaction conditions (room temperature, no need for strong acids or alkalis or high temperatures), environmental friendliness, and low cost, providing a feasible solution for the biodegradation of polyurethane waste.

[0078] When applied to environmental protection and waste treatment, it can take the form of concentrated enzyme solution, powder formulation, immobilized enzyme particles, bioreactor packing material, or bio-enhanced compost additive. Applying it to the biodegradation process of polyurethane waste (especially types that are difficult to physically recycle or chemically treat) can significantly accelerate the decomposition rate, converting it into smaller molecules. This enables efficient biological recycling or environmentally friendly disposal of polyurethane waste, providing a green biotechnology solution for addressing stubborn plastic components in "white pollution," and has significant environmental benefits.

[0079] When applied to industrial cleaning and material surface treatment, its formulation can be concentrated cleaning solutions, enzyme-based gel cleaners, spray stain removers, or immersion treatment solutions. It is used for cleaning industrial equipment containing polyurethane residues (such as pipes, molds, and spraying equipment), removing stubborn polyurethane coatings / adhesive stains from instrument or tool surfaces, or for pre-treatment of polyurethane impurities on the surfaces of specific materials (such as recycled textiles). Compared to strong chemical cleaners, enzymatic treatment offers advantages such as mild conditions (normal temperature and pressure), high selectivity, low corrosivity, and environmental friendliness, effectively improving cleaning efficiency and material treatment quality.

[0080] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A cutinase ChCutl having the ability to degrade polyurethane plastics, characterized in that, The amino acid sequence of the cutinase is shown as SEQ ID NO.

1.

2. A gene encoding cutinase ChCutl having the ability to degrade polyurethane plastics, characterized in that, The nucleotide sequence of the encoding gene is shown as SEQ ID NO.

2.

3. A method for extracellular expression of a cutinase ChCutl having the ability to degrade polyurethane plastic, characterized in that, The steps are as follows: (a) providing an extracellular signal peptide sequence, the amino acid sequence of which is SEQ ID NO. 3, encoding 89 amino acids, with a theoretical molecular weight of 9.3 kDa; (b) cloning the complete cutinase gene ChCut1 (SEQ ID NO: 2) from Cladosporium halotolerans 6UPA1 and the above signal peptide into the expression vector pGADHL, retaining its natural structure, to construct a recombinant plasmid; (c) using yeast as the host, introducing the recombinant plasmid by lithium acetate transformation; (d) screening the transformants using SD-Leu auxotrophic medium and inducing expression; (e) obtaining the cutinase from the crude enzyme solution of the yeast fermentation broth.

4. The extracellular expression method according to claim 3, wherein The yeast is Saccharomyces cerevisiae.

5. The cutinase ChCut1 of claim 1 is applied in one or both of the following 1) and 2): 1) polyurethane waste degradation; 2) industrial cleaning and material surface treatment.

6. The application of claim 5, wherein the industrial cleaning and material surface treatment includes cleaning industrial equipment containing polyurethane residues, removing stubborn polyurethane coatings / stains from the surface of equipment or tools, or pretreatment of surface polyurethane impurities for specific materials.

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