Fungus cutinase CpLCt1 capable of degrading polyester plastics and application of fungus cutinase CpLCt1

Abstract

The invention discloses fungal cutinase CpLCt1 capable of degrading polyester plastics and application of the fungal cutinase CpLCt1. The invention provides a cutinase coding gene capable of degrading polyurethane plastic, the nucleotide sequence of the cutinase coding gene is SEQ ID NO.1, and the amino acid sequence of the coded cutinase protein is SEQ ID NO.2. The recombinant cutinase obtained by using an engineering strain constructed by the gene takes 4-nitrobenzene butyrate as a substrate, and the specific enzyme activity can reach 52.94 U / mg through determination. The cutinase can destroy the structure of polyurethane plastic, and an obvious transparent hydrolysis ring is generated on a flat plate. The cutinase can degrade PUR foam, the shape of the PU foam can be obviously changed within 3 days at the temperature of 55 DEG C, and the PU foam is in a broken powder shape. Therefore, the cutinase coding gene can be widely applied to degradation and resource utilization of waste polyester plastics as an element.

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 worth 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, restricting 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 the pollution of waste plastics.

[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 and 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 interface of PU plastics, the research on the biodegradation of PU plastics faces great challenges. At present, researchers have identified a large number of polyester plastic-degrading microorganisms, including Cladosporium, Bacillus, Pseudomonas, Cryptococcus, Monospora and Staphylococcus, which secrete various hydrolases that can degrade PU or PBAT. However, most of the microorganisms and enzymes discovered so far have shown limited efficiency in degrading PU plastics. Therefore, the exploration of plastic-degrading enzymes with PU degradation ability is of great significance for the disposal and resource utilization of PU waste 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 of which is: 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 biodegradation of polyester plastics.

[0017] The cutinase CpLCut1 of the present invention can degrade PU foam and PU plastic synthetic oligomer PBA-PU. At 55°C, within 3 days, the PU foam treated with cutinase undergoes a significant change in morphology and appears as a broken powder. At 37°C, the enzyme solution is added dropwise to a PBA plate and cultured overnight in an incubator. After being degraded by the PU hydrolase, a transparent circle appears on the plate.

[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 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 uses Cladosporium sp.L-5 (deposit number GDMCCNo: 62786, disclosed in ZL202310117342.0) as material, refers to genome sequence information and combines PCR amplification to successfully obtain the cutinase gene sequence. The full length of the gene (from the start codon to the stop codon) is 663bp, the G+C content is 58.67%, and it encodes 221 amino acids, and the first 16 amino acids at the N-terminus are signal peptides.

[0023] 2. The recombinant cutinase obtained by the engineered strain constructed by using the gene takes 4-nitrophenylbutyrate as substrate, and the specific enzyme activity can reach 52.94U / mg. The cutinase can destroy the structure of polyurethane plastics and produce obvious transparent hydrolysis circles on the plate. The cutinase can degrade PUR foam. At 55°C, it can be clearly observed that the PU foam morphology changes significantly within 3 hours, showing a broken powder state.

[0024] 3. The plastic bioenzymatic depolymerization technology of the present invention has mild conditions, efficient process, few by-products, and is green and environmentally friendly. It is an ideal means for the disposal of waste plastics and a research hotspot at home and abroad. It has important 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 amplification 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 on the hydrolysis of PBA and degradation of PU foam by cutinase CpLCut1 (A) Identification of PBA hydrolysis zones on a flat plate (1: CpLCut1 drop site; 2: CpLCut2 drop site; 3: Tris-Hcl drop site; 4: CpLCut1 drop site treated in boiling water bath); (B) PU foam degradation experiment (the left side is the blank control, the right side is the degradation of PU foam by CpLCut1 crude enzyme solution for 3 days) DETAILED DESCRIPTION

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

[0030] Referring to the Cladosporium sp.L-5 genome information in the NCBI database (the deposit number is 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 IDNO.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 Escherichia coli strain and its plasmid were used as templates for subsequent protein expression. During the heterologous expression of the protein, the N-terminal signal peptide was removed. The primers used for its expression in Pichia pastoris are F1 and R1. The PCR amplification results are shown in 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 connected to the pEFαA vector by enzyme ligation to construct the pEFαA-CpLCut1 plasmid. After sequencing verification, the constructed plasmid was linearized using the restriction endonuclease Nde I to linearize the correct recombinant plasmid. The linearized plasmid was introduced into Pichia pastoris GS115 competent cells by electroporation and spread on a YPD plate containing 100 μg / mL Zeocin. After growth, colony PCR was performed using the characteristic primers of the target gene to detect whether the target gene was integrated into the yeast chromosome. The positive clone was named P. pastoris GS115 (pEFαA-CpLCut1). The expression strain P. pastoris GS115 (pEFαA-CpLCut1) was streaked on a plate, and a single colony was picked up and placed in a 50 mL liquid YPD flask, and cultured at 30°C, 200 rpm for 24 h; then centrifuged at room temperature at 4000 rpm for 5 min, the supernatant was discarded, and the bacteria were resuspended in 25 mL BMMY medium to begin inducing yeast cell expression; culture was continued at 30°C, 200 rpm, and methanol was added every 24 h to a final concentration of 0.5% (v / v), and cultured for a total of 96 h; after the culture was completed, the collected supernatant was taken for SDS-PAGE electrophoresis, and a shallow but single protein band was found between 17-25 kDa indicated by the protein marker, and its size 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 the induction expression is completed is subjected to Ni2+-NTA affinity chromatography, and the steps are as follows: the Ni2+-NTA affinity chromatography column is cleaned with 20% ethanol, deionized water, 0.5M NaOH, and deionized water; the column is then washed twice with 20mM Tris-HCl (pH 7.6) buffer to balance the column; the crude enzyme solution (i.e., the culture supernatant) is added and incubated for 30 minutes to allow the target protein with His-Tag to hang on the column; the centrifuge tube is placed on ice to collect the crude enzyme solution, i.e., the pass-through solution, and the pass-through solution can be re-passed through the column to increase the protein amount; the column is then washed twice with 20mM Tris-HCl (pH 7.6) buffer to collect the eluate; the prepared 20mM, 50mM, 100mM, 200mM, and 300mM imidazole solutions are added to the column from low to high concentrations to elute the protein, the protein solution is collected on ice, and the labeling is done.

[0036] After the first affinity chromatography purification of the protein, the optimal imidazole elution concentration is determined based on the protein concentration measured in different imidazole eluates, and this concentration is directly used in subsequent protein purification to obtain the maximum amount of target protein. After purification, the imidazole in the collected solution is removed by dialysis.

[0037] Next, Ni2+-NTA affinity chromatography was used to obtain purified CpLCut1 enzyme solution. The reaction system was set up to 1 mL: 10 μL 10 mM 4-nitrophenylbutyrate (PNB), 10 μL enzyme solution, and 980 μL 50 mM PBS (pH 7.2-7.4). After the preparation, it was placed in a 37°C water bath for 10 minutes, and then the reaction solution was added to a 96-well plate and the OD was measured using an ELISA reader. 410 The enzyme activity unit is defined as: under standard conditions, the amount of enzyme μL required to catalyze the production of 1 μmol of p-nitrophenol in 1 min is defined as 1 enzyme activity unit U. The protein concentration in the enzyme solution and its esterase activity were determined in turn according to the Brandford method and the p-nitrophenol method. The protein concentration of CpLCut1 in the purified enzyme solution was determined to be 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 10 mM various substrates, 10 μL enzyme solution, 980 μL PBS buffer (pH 8.0)), and the enzyme activity was measured after reacting at 55°C or 65°C for 10 minutes. The enzyme activity of the recombinant enzyme was determined to hydrolyze esters of different chain lengths. The enzyme activity of the recombinant enzyme when no enzyme solution was added and all PNB was naturally hydrolyzed to PNP was set as 100%, and the percentage of the recombinant enzyme hydrolyzing the substrate was calculated as the relative enzyme activity. The results showed that the recombinant enzyme CpLCut1 had a significant degradation ability for substrates with shorter chain lengths such as C2, C4, and C6 ( Figure 3 ).

[0040] Example 4 Determination of the ability of the recombinant enzyme CpLCut1 to hydrolyze polyurethane plastics

[0041] The purified recombinant CpLCut1 enzyme was added to the prepared PBA plate according to the recombinant protein SDS-PAGE identification and PBA plate hydrolysis circle identification method, and placed in a 37°C incubator overnight. Then the PBA plate hydrolysis circle method was used, that is, 3 Oxford cups were placed on the PBA plate, 200μL enzyme solution, 200μL boiling water bath 10min inactivated enzyme solution, and 200μL Tris-HCl (pH 7.6) were added to them respectively; the plate was placed in a 37°C constant temperature incubator for 12 to 48 hours, and then the plate was checked for transparent circles. The results showed that the PBA plate was opaque milky white in its original state, and after being degraded by PU hydrolase, transparent circles appeared in and around the Oxford cup, indicating that CpLCut1 had 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, it was found that the PU foam in the experimental group had a significant change in morphology and appeared as a broken powder, which also shows that CpLCut1 has the ability to degrade 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 bacteria.

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.

8. Use of the cutinase CpLCut1 according to claim 1 in the biodegradation of polyester plastics.

9. The use according to claim 8, characterized in that: The polyester plastic is polyurethane plastic, preferably PU foam or PU plastic synthetic oligomer PBA-PU.

Images