A fungal cutinase CpLCut1 capable of degrading polyester plastics and its application

Abstract

The present invention discloses a fungal cutinase CpLCut1 capable of degrading polyester plastics and its application. The present invention provides a cutinase encoding gene capable of degrading polyurethane plastics, the nucleotide sequence of which is SEQ ID NO.1, and the amino acid sequence of the encoded cutinase protein is SEQ ID NO.2. The recombinant cutinase obtained by the engineered strain constructed using the gene uses 4-nitrophenylbutyrate as a substrate, and the specific enzyme activity can reach 52.94 U / mg. The cutinase can destroy the structure of polyurethane plastics and produce a clear transparent hydrolysis zone on a flat plate. The cutinase can degrade PUR foam, and a significant change in the morphology of the PU foam can be clearly observed within 3 hours at 55°C, presenting a broken powder. Therefore, the use of the cutinase encoding gene as an element can be widely applied to the degradation and resource utilization of waste polyester plastics.

Description

Technical Field

[0001] The invention belongs to the fields of environmental science and biotechnology, and relates to a fungal cutinase CpLCut1 capable of degrading polyester plastic and application thereof. Background Art

[0002] Polyurethane (PU), whose full name is polyurethane, is a high-molecular organic compound synthesized from polyols and polyisocyanates. In 2020, the global PU market size was valued at US$70.67 billion, and PU production accounted for 6% of plastics. It has become the second largest polyester plastic in the world and is widely used in textiles, construction, building materials, automobiles, national defense and other fields. However, the irrational disposal of petroleum-based plastics represented by waste polyurethane (PU) has caused serious environmental pollution and waste of carbon resources, which has restricted the development of my country's green economy. At present, the treatment of waste plastics mainly relies on physical and chemical recycling or incineration and landfill, which does not meet the national strategic needs of "same-level plastic waste and high-value-added utilization". Therefore, with the development needs of green biomanufacturing in my country, plastic biodepolymerization has become an important means to solve waste plastic pollution.

[0003] In recent years, breakthroughs have been made in the biodepolymerization of PET plastics. The French company CARBIOS has established a demonstration line that can process 30,000 tons of PET waste plastics annually, proving that biodepolymerization recycling technology is an ideal way to recycle waste plastic resources in the future.

[0004] However, due to the complex structural components and highly hydrophobic interfaces of PU plastics, research on their biodegradation presents significant challenges. Currently, researchers have identified a large number of polyester plastic-degrading microorganisms, including Cladosporium, Bacillus, Pseudomonas, Cryptococcus, Monosporus, and Staphylococcus. These microorganisms secrete various hydrolases capable of degrading PU or PBAT. However, most of the microorganisms and enzymes discovered to date have shown limited efficiency in degrading PU plastics. Therefore, the discovery of plastic-degrading enzymes with PU degradation capabilities is of great significance for the disposal and resource utilization of waste PU plastics. Summary of the Invention

[0005] The purpose of the present invention is to provide a cutinase capable of degrading polyurethane foam plastics and a coding gene thereof.

[0006] Another object of the present invention is to provide the application of the cutinase or gene.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A cutinase CpLCut1, the amino acid sequence of which is shown in SEQ ID NO.2, encodes 221 amino acids, has a theoretical molecular weight of 22.41 kDa, and an isoelectric point of 7.81. The first 16 amino acids at the N-terminus of the protein are a signal peptide sequence, and the amino acid sequence is shown in SEQ ID NO.3.

[0009] The cutinase is derived from Cladosporium sp. L-5.

[0010] The coding gene of the cutinase CpLCut1.

[0011] The nucleotide sequence of the cutinase gene of the present invention is: SEQ ID NO. 1. The full length of the gene (from the start codon to the stop codon) is 663 bp, and the G+C content is 58.67%.

[0012] A recombinant expression plasmid containing the cutinase CpLCut1 encoding gene.

[0013] A recombinant microorganism containing the recombinant plasmid of the present invention.

[0014] The recombinant microorganism preferably uses Pichia pastoris as a host strain.

[0015] The cutinase gene, recombinant plasmid and recombinant microorganism of the present invention are used in genetic engineering for biodegradation of polyester plastics.

[0016] Application of the cutinase CpLCut1 of the present invention in the biodegradation of polyester plastics.

[0017] The cutinase CpLCut1 described in this invention can degrade PU foam and the synthetic oligomer PBA-PU of PU plastic. Within three days at 55°C, the PU foam treated with cutinase undergoes a significant morphological change, becoming a crushed powder. When the enzyme solution is added dropwise to a PBA plate and incubated overnight at 37°C in an incubator, a transparent ring appears on the plate after degradation by the PU hydrolase.

[0018] The cutinase of the present invention degrades polyurethane plastics, and can degrade PU foam and PU plastic synthetic oligomer PBA-PU.

[0019] The cutinase of the present invention is used in the production and application of polyurethane plastic degradation, conversion and resource utilization.

[0020] The cutinase gene of the present invention is used as a degradation element in the construction of engineered chassis cells.

[0021] Beneficial effects

[0022] 1. The present invention successfully obtained the cutinase gene sequence from Cladosporium sp. L-5 (GDMCC No. 62786, disclosed in ZL202310117342.0) by referring to the genome sequence information and combining PCR amplification. The full length of the gene (from the start codon to the stop codon) is 663 base pairs, with a G+C content of 58.67%, encoding 221 amino acids, with the first 16 amino acids at the N-terminus serving as a signal peptide.

[0023] 2. The recombinant cutinase obtained from the engineered strain constructed using this gene exhibited a specific enzyme activity of 52.94 U / mg using 4-nitrophenylbutyrate as a substrate. This cutinase is capable of destroying the structure of polyurethane plastics, producing distinct, transparent hydrolysis zones on a flat plate. This cutinase is also capable of degrading polyurethane foam. Within three hours at 55°C, the morphology of the foam was significantly altered, resulting in a crushed, powdery state.

[0024] 3. The enzymatic depolymerization technology of this invention offers mild conditions, high efficiency, few byproducts, and is environmentally friendly. It is an ideal method for waste plastic disposal and a research hotspot both domestically and internationally. It has significant application value in the disposal and resource utilization of waste PU plastics. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 PCR electrophoresis of CpLCut1 gene (M: DL10,000 DNA Marker; 1: CpLCut1 amplified product)

[0026] Figure 2 SDS-PAGE electrophoresis of recombinant cutinase CpLCut1

[0027] Figure 3 Degradation ability of recombinant enzyme CpLCut1 on different substrates

[0028] Figure 4 Observation of the hydrolysis of PBA and degradation of PU foam by cutinase CpLCut1 (A) Identification of PBA hydrolysis zones on a flat plate (1: CpLCut1 addition site; 2: CpLCut2 addition site; 3: Tris-HCl addition site; 4: CpLCut1 addition site treated in a boiling water bath); (B) PU foam degradation experiment (left: blank control; right: PU foam degradation by CpLCut1 crude enzyme solution after 3 days) DETAILED DESCRIPTION

[0029] Example 1 PCR amplification of the gene encoding cutinase CpLCut1

[0030] With reference to the Cladosporium sp.L-5 genome information in the NCBI database (deposit number GDMCCNo: 62786, disclosed in ZL202310117342.0) and combined with ORF prediction, the full-length sequence of the target protein encoding gene was obtained. The full length of the gene (from the start codon to the stop codon) is 663bp, the G+C content is 58.67%, the gene sequence is SEQ ID NO.1, encoding 221 amino acids, the first 16 amino acids at the N-terminus are a signal peptide, and its amino acid sequence is SEQ ID NO.2. Nanjing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the full sequence, connect it to the pET29a plasmid, and transform it into Escherichia coli DH5α. The E. coli strain and its plasmid served as templates for subsequent protein expression. During the heterologous expression of the protein, the N-terminal signal peptide was removed, and the primers used for its expression in Pichia pastoris were F1 and R1. The PCR amplification results are shown in Figure 2. Figure 1 .

[0031] F1: 5-CGGCCGTCTCGGATCGGTACCATGAAGTTCACTACAGCCGTTGC-3 (Kpn I); R1: 5-GAGATGAGTTTTTGTTCTAGAUUACACCACCACCACCACGATGCCACCAGCAGCAGC-3 (Xba I).

[0032] Example 2 Heterologous Expression of Recombinase CpLCut1 in Pichia pastoris

[0033] Using Pichia pastoris as the expression host, the PCR amplification product of the CpLCut1 cutinase-encoding gene was ligated into the pEFαA vector via enzyme ligation to construct the pEFαA-CpLCut1 plasmid. After sequencing verification, the constructed plasmid was linearized using the restriction endonuclease Nde I. The linearized plasmid was introduced into Pichia pastoris GS115 competent cells by electroporation and plated onto YPD plates containing 100 μg / mL Zeocin. After growth, colony PCR was performed using primers specific for the target gene to verify integration into the yeast chromosome. Positive clones were designated P. pastoris GS115 (pEFαA-CpLCut1). The expression strain P. pastoris GS115 (pEFαA-CpLCut1) was streaked onto a plate, and a single colony was picked and placed in a 50 mL YPD flask. The flask was cultured at 30°C and 200 rpm for 24 h. The culture was then centrifuged at 4000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 25 mL BMMY medium to begin inducing yeast cell expression. The culture was continued at 30°C and 200 rpm, with methanol added every 24 h to a final concentration of 0.5% (v / v) for a total of 96 h. After the culture was completed, the collected supernatant was subjected to SDS-PAGE electrophoresis, which revealed a faint but single protein band between 17 and 25 kDa indicated by the protein marker, which was consistent with the theoretical size of CpLCut1 of 22.4 kDa ( Figure 2 ).

[0034] Example 2 Activity determination of recombinase CpLCut1 in Pichia pastoris

[0035] The CpLCut1 protein induced and expressed by P. pastoris GS115 was purified. First, the culture supernatant after induced expression is subjected to Ni2+-NTA affinity chromatography, and the steps are as follows: wash the Ni2+-NTA affinity chromatography column with 20% ethanol, deionized water, 0.5M NaOH, and deionized water; then wash the column twice with 20mM Tris-HCl (pH 7.6) buffer to balance the column; add crude enzyme solution (i.e., culture supernatant) and incubate for 30 minutes to allow the target protein with His-Tag to hang on the column; place the centrifuge tube on ice and collect the crude enzyme solution, i.e., the flow-through. To increase the protein amount, the flow-through can be re-passed through the column; then wash twice with 20mM Tris-HCl (pH 7.6) buffer and collect the eluate; add the prepared 20mM, 50mM, 100mM, 200mM, and 300mM imidazole solutions from low to high concentrations to elute the protein, collect the protein solution on ice, and mark it.

[0036] After the first affinity chromatography purification of the protein, determine the optimal imidazole elution concentration based on the protein concentration measured in different imidazole eluates. Use this concentration directly in subsequent protein purification to obtain the maximum amount of target protein. After purification, use dialysis to remove the imidazole from the collected solution.

[0037] Next, purified CpLCut1 enzyme solution was obtained using Ni2+-NTA affinity chromatography. A 1 mL reaction system was set up: 10 μL 10 mM 4-nitrophenylbutyrate (PNB), 10 μL enzyme solution, and 980 μL 50 mM PBS (pH 7.2-7.4). After the mixture was prepared, it was incubated in a 37°C water bath for 10 minutes. The reaction solution was then added to a 96-well plate and the OD was measured using a microplate reader. 410 One unit of enzyme activity is defined as the amount of enzyme (μL) required to catalyze the production of 1 μmol of p-nitrophenol in 1 minute under standard conditions. Protein concentration and esterase activity in the enzyme solution were determined using the Brandford and p-nitrophenol methods, respectively. The protein concentration of CpLCut1 in the purified enzyme solution was approximately 0.053 mg / mL, and the specific enzyme activity was 52.94 U / mg.

[0038] Example 3 Analysis of the hydrolysis substrate spectrum of the recombinant enzyme CpLCut1

[0039] The CpLCut1 recombinant enzyme was added to a reaction system containing various ester substrates (10 μL of 10 mM substrates, 10 μL of enzyme solution, 980 μL of PBS buffer (pH 8.0)). After reacting at 55°C or 65°C for 10 minutes, the enzyme activity was measured to determine its ability to hydrolyze esters of different chain lengths. The activity in which all PNBs were naturally hydrolyzed to PNPs without the addition of enzyme solution was set as 100%, and the percentage of substrate hydrolysis by the recombinant enzyme was calculated as the relative enzyme activity. The results showed that the recombinant enzyme CpLCut1 had a significant ability to degrade substrates with shorter chain lengths, such as C2, C4, and C6 ( Figure 3 ).

[0040] Example 4 Determination of the Hydrolysis Ability of Recombinant Enzyme CpLCut1 on Polyurethane Plastics

[0041] The purified recombinant CpLCut1 enzyme was added dropwise to a pre-prepared poly(vinyl alcohol) (PBA) plate according to the recombinant protein SDS-PAGE and PBA plate hydrolysis zone assays. The plate was then incubated overnight at 37°C. The PBA plate hydrolysis zone assay was then performed. Three Oxford cups were placed on the PBA plate, and 200 μL of enzyme solution, 200 μL of enzyme inactivation solution (inactivated by boiling water for 10 minutes), and 200 μL of Tris-HCl (pH 7.6) were added to each of the three Oxford cups. The plates were incubated at 37°C for 12 to 48 hours and then examined for the formation of a clearing zone. The results showed that the PBA plate was opaque, milky white in its original state. However, after degradation by the PU hydrolase, a clearing zone appeared around the Oxford cup and its surrounding area, indicating that CpLCut1 possesses PU hydrolase activity. In addition, after the crude enzyme solution of CpLCut1 (containing about 0.3-0.5 mg protein) was used to degrade PU foam plastic at 55°C for 3 days, the PU foam in the experimental group changed significantly and appeared as a broken powder, which also shows the degradation ability of CpLCut1 on PU plastic ( Figure 4 ).

Claims

1. A cutinase CpLCut1, characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the cutinase CpLCut1 according to claim 1.

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

1.

4. A recombinant expression plasmid containing the coding gene according to claim 2 or 3.

5. A recombinant microorganism containing the coding gene according to claim 2 or 3 or the recombinant expression plasmid according to claim 4.

6. The recombinant microorganism according to claim 5, characterized in that Pichia pastoris was used as the host strain.

7. Genetic engineering application of the coding gene according to claim 2 or 3, the recombinant expression plasmid according to claim 4, or the recombinant microorganism according to claim 5 or 6 in biodegradable polyester plastics, wherein the polyester plastic is PU foam or PU plastic synthetic oligomer PBA-PU.

8. Use of the cutinase CpLCut1 according to claim 1 in the biodegradation of polyester plastics, wherein the polyester plastics are PU foams or PU plastic synthetic oligomers PBA-PU.

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