A cutinase for degradable polyester plastics and use thereof

Abstract

The application discloses a cutinase capable of degrading polyester plastic and application thereof. The application provides a cutinase gene capable of degrading polyester plastic, wherein the nucleotide sequence of the cutinase gene is SEQ ID NO. 1, and the amino acid sequence of the coded cutinase protein is SEQ ID NO. 2. The cutinase can destroy the complete structure of plastic, and the surface of the plastic appears holes, cracks and other erosion. In addition, the cutinase can degrade different types of polyester plastic, wherein the weight loss rates of PUR foam and polyester plastic PCL reach 33.88% and 83.44% respectively within 2 days, and the degradation rates of agricultural degradable mulch PBAT and PU plastic synthetic oligomer PBA-PU reach 66.70% and 64.41% respectively within 4 days. The cutinase gene can be widely applied to degradation and resource utilization of waste polyester plastic as an element.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental science and biotechnology, and relates to a cutinase capable of degrading polyester plastic and application thereof. BACKGROUND

[0002] Polyurethane (PU) plastic is a polymer condensed by polyisocyanate and polyol, etc. PU products are rich in types and occupy a large market in the world, and are widely used in the fields of textiles, construction, building materials, automobiles, national defense, etc. PU has become the second largest polyester plastic in the world, with 1470 million tons produced in China in 2020 and 1175 million tons consumed. However, a large amount of used PU plastics are discarded in the natural environment, which not only causes waste of carbon resources, but also causes an increasingly serious ecological disaster. At present, the main methods for plastic waste are landfill, incineration, mechanical recycling and chemical recycling. However, these disposal methods can cause potential environmental pollution. In addition, PU plastics have complex structures and diverse types, and are difficult to be effectively reused by existing physical and chemical recycling methods.

[0003] Compared with traditional treatment methods, the use of microorganisms or enzymes for biological recycling of plastics is considered to be one of the most promising methods. At present, the establishment of PET plastic enzymatic recycling technology system based on high-efficiency PET depolymerase has made the enzymatic degradation and product recovery of waste plastics become the focus of attention. The biological recycling of polyethylene terephthalate (PET) plastic has entered the pre-industrialization stage. The French Carbios company uses the cutinase LCC from plant compost to decompose 97% of PET plastic in 16 hours, and the monomers are repolymerized to form new plastics.

[0004] However, there are few depolymerase elements for degradable PU plastics, and the current research is still in the stage of resource mining. The existing reports include a new carbamate hydrolase for enzymatic decomposition of polyurethane (CN201980041682.1), cutinase BC-CUT-013 and Thc_Cut1 (CN202180069579.5) capable of degrading polyurethane PU, and complete depolymerization of thermoplastic polyester polyurethane plastic by combining PETase and carbamate enzyme Aes72 (CN202310054434.9). In addition, some cutinases, esterases and / or lipases also have certain ability to degrade PU plastics, as mentioned in the relevant literature. However, the complex structure, high crystallinity and high hydrophobicity of PU plastics directly limit the degradation of enzymes. Therefore, not all esterases have the ability to degrade plastics. Therefore, it is of great significance to mine plastic-degrading enzymes with PU degradation ability for the disposal and resource utilization of PU waste plastics. SUMMARY

[0005] The present application aims to provide a cutinase and its encoding gene which can degrade polyurethane foam and plastic such as agricultural mulch PBAT, and the enzyme can be identified by amino acid sequence motif (primary structure), secondary structure element, and tertiary structure element and amino acid sequence online BLAST comparison. The cutinase is derived from Blastobotrys.

[0006] Another object of the present application is to provide a genetically engineered bacterium containing the cutinase gene.

[0007] Still another object of the present application is to provide the application of the gene and the protein encoded by the gene.

[0008] The cutinase gene of the present application has a nucleotide sequence of SEQ ID NO. 1, and the full length (from the start codon to the stop codon) of the gene is 669 bp, and the G+C content is 54.26%; the cutinase protein encoded by the cutinase gene has an amino acid sequence of SEQ ID NO. 2, encodes 222 amino acids, and has a theoretical molecular weight of 23.39 KD and an isoelectric point of 4.65; the N-terminal first 18 amino acids of the protein are signal peptide sequences, and the amino acid sequence is SEQ ID NO. 3.

[0009] The recombinant microorganism containing the recombinant plasmid of the present application.

[0010] The recombinant microorganism, preferably Escherichia coli and Pichia pastoris as host bacteria.

[0011] The cutinase of the present application has a specific enzyme activity of 771.8 U / mg with 4-nitrophenyl butyrate as substrate, at 37℃ and pH 7.

[0012] The cutinase gene, recombinant plasmid, and recombinant microorganism of the present application are genetically engineered for application in polyester plastic biodegradation.

[0013] As a preferred embodiment of the present application, the polyester plastic includes but is not limited to PUR foam, agricultural mulch PBAT, degradable plastic PCL, and PL.

[0014] The cutinase BaCut1 of the present application is used in polyester plastic biodegradation.

[0015] As a preferred embodiment of the present application, the polyester plastic includes but is not limited to PUR foam, agricultural mulch PBAT, degradable plastic PCL, and PL.

[0016] The cutinase BaCut1 can degrade PUR foam, agricultural degradable mulch PBAT, PU plastic synthetic oligomer PBA-PU and polyester plastic PCL, and the weight loss rates of PUR foam and PCL are 33.88% and 83.44% respectively within 2 days at 37 DEG C, and the degradation rates of PBAT and PBA-PU are 66.70% and 64.41% respectively within 4 days at 37 DEG C.

[0017] The cutinase of the application can degrade polyurethane plastic, and can degrade PUR foam and PU plastic synthetic oligomer PBA-PU to produce adipic acid.

[0018] The cutinase of the application can be used in the production and application of polyurethane plastic degradation, conversion and resource utilization.

[0019] The cutinase gene of the application can be used as a degradation element in the construction of an engineered chassis cell.

[0020] Beneficial effects

[0021] 1. The yeast strain screened from soil samples is used as the material, and the cutinase gene sequence is successfully obtained by referring to the genomic sequence information and combining PCR amplification. The full length (from the start codon to the stop codon) of the gene is 669 bp, the G+C content is 54.26%, 222 amino acids are encoded, and the first 18 amino acids at the N terminal are signal peptides.

[0022] 2. The product expressed by the cutinase gene has high degradation capacity for PUR foam, agricultural mulch PBAT, degradable plastic PCL and PU plastic synthetic oligomer PBA-PU, etc., when the enzyme concentration is 40 μg / mL and the substrate concentration is 8 mg / mL.

[0023] 3. The plastic biocatalytic depolymerization technology of the application has mild technical conditions, high process efficiency, less by-products and green environmental protection, and is an ideal means for disposal of waste plastics and a research hotspot at home and abroad. The application has important application value in the disposal and resource utilization of waste PUR plastic and agricultural mulch. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 PCR amplification electrophoretogram of cutinase coding gene

[0025] Figure 2 SDS-PAGE electrophoretogram of recombinant cutinase BaCut1

[0026] M: protein marker; 1: purified recombinant protein of host cell broken supernatant without target gene; 2: host cell broken supernatant containing target gene; 3: purified recombinant protein

[0027] Figure 3TEM observation of cutinase BaCut1 degrading PUR foam plastic and PBAT film

[0028] Figure 4 Weight loss of different types of polyester plastics before and after degradation by cutinase BaCut1 DETAILED DESCRIPTION

[0029] Example 1 PCR amplification of cutinase encoding gene

[0030] The full-length sequence of the target protein encoding gene was obtained by referring to the Blastobotrys sp. G-9 genome information (PRJNA1061465) in the NCBI database and combining ORF prediction. The full-length of the gene (from the start codon to the stop codon) is 669 bp, the G+C content is 54.26%, the gene sequence is SEQ ID NO. 1, it encodes 222 amino acids, and the first 18 amino acids at the N terminus are a signal peptide, and the amino acid sequence is SEQ ID NO. 2. Nanjing Qikexing Biological Technology Co., Ltd. was commissioned to synthesize the full sequence, which was connected to the pET29a plasmid and transformed into Escherichia coli DH5α. The E. coli strain and its plasmid were used as templates for subsequent protein expression. In the process of heterologous expression of the protein, the N-terminal signal peptide was removed, and the primers used for expression in E. coli were F1 and R1, and the primers used for expression in Pichia pastoris were F2 and R2, and the PCR amplification results are shown in Figure 1 .

[0031] F1: 5-gctgatatcggatccgaattcATGGCTCCCCTGGAGCGA-3 (EcoR I);

[0032] R1: 5-gtggtggtggtggtgctcgagTTAGGAAGTCAAGGCCTTAACGA-3 (Xho I);

[0033] F2: 5-cggccgtctcggatcggtaccATGGCTCCCCTGGAGCGA-3 (Kpn I);

[0034] R2: 5-gagatgagtttttgttctagaTCAatgatgatgatgatgatgGGAAGTCAAGGCCTTAACG-3 (Xba I).

[0035] Example 2 Expression, purification and activity determination of cutinase in E. coli

[0036] The PCR amplification product of the cutinase gene without signal peptide was connected to pET29a vector by enzyme ligation to construct pET29a-baCutl plasmid. The plasmid was introduced into E. coli BL21 (DE3) competent cells, and the cells were coated on LB plates containing 50 mg / L kanamycin. Single colonies were selected and verified by sequencing to obtain E. coli cells containing pET29a-baCutl plasmid. The constructed E. coli containing expression plasmid was induced by IPTG to induce the expression of recombinant protein in E. coli. The recombinant protein was purified by Ni column affinity chromatography. The results showed that the cutinase had a good expression amount in E. coli, reaching 200.73 mg / L, and the heterologous expression of recombinant cutinase with high purity was obtained by Ni column purification. Figure 2 The activity of the cutinase was determined and the degradation performance was evaluated. The specific enzyme activity of the cutinase reached 771.8 U / mg under the condition of 4-nitrophenyl butyrate as substrate, 37°C and pH 7.

[0037] Example 3 Expression and activity determination of cutinase in Pichia pastoris

[0038] The PCR amplification product of the cutinase gene without signal peptide was connected to pEFaA vector by enzyme ligation to construct pEFaA-baCutl plasmid. After sequencing verification, the constructed plasmid was linearized by restriction enzyme Sca I. The linearized plasmid was introduced into Pichia pastoris GS115 competent cells by electroporation method, and coated on YPD plates 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 (pEFaA-baCutl). The expression strain P. pastoris GS115 (pEFaA-baCutl) was streaked and cultured, and single colonies were picked into 50 mL liquid YPD flask and cultured at 28°C, 200 rpm for 24 h. Then, the cells were centrifuged at 4000 rpm for 5 min at room temperature, and the supernatant was discarded. The cells were resuspended with 25 mL BMMY medium, and the expression of the yeast cells was induced. The culture was continued at 28°C, 200 rpm, and methanol was added every 24 h to a final concentration of 0.5% (v / v), and the culture was continued for 96 h. After the culture was completed, the expression amount of the target protein was detected by SDS-PAGE, and the expression amount was 54.55 mg / L.

[0039] Example 4 Determination of the degradation ability of cutinase BaCutl to polyester plastics

[0040] The 40 mg PUR foam, agricultural degradable mulch PBAT, PU plastic synthetic oligomer PBA-PU, and polyester degradable PCL were weighed. The PUR foam was sterilized by steam sterilization at 121°C for 20 min, the agricultural degradable mulch PBAT was sterilized by long-term (more than 3 days) immersion in 75% ethanol, and the PBA-PU and PCL were sterilized by immersion in 75% ethanol for 20 min and ultraviolet sterilization for 30 min. Then the sterile and dry plastics were incubated with 200 μg of BaCut1 (5 mL, 50 mM Tris-HCl, pH 8). After incubation at 37°C for a period of time, the plastics were filtered with weighed filter paper, washed with deionized water 3 times to remove the residual plastics on the filter paper, then placed in a 40°C drying oven to dry until the mass did not change, then the sample mass was weighed, and the mass of the remaining plastics was obtained by subtracting the mass of the filter paper. The weight loss rate of the plastics was calculated by the ratio of the weight loss of the plastics to the initial weight of the plastics. The protein solution precipitated by 0.4 M TCA and Tris-HCl buffer (50 mM, pH 8) were used as controls. The BaCut1-treated plastics were dried in an oven, cut into thin slices about 0.20 mm thick with a clean knife blade, fixed on the sample stage of the scanning electron microscope with black double-sided tape, and coated with a layer of gold film on the surface of the plastic slices with an ion sputtering instrument. Then the sample stage was fixed on the sample seat and pushed into the scanning electron microscope SU8010 (Hitachi, Japan). The surface morphology of the PUR plastics was observed and photographed by adjusting the magnification, focal length, and observation position.

[0041] The results of electron microscopy observation of plastics in different treatment groups showed that the surfaces of PUR foam and PBAT plastics treated with cutinase BaCut1 were eroded and presented as fragments. Figure 3 The results of plastic weight loss showed that cutinase BaCut1 could degrade different types of polyester plastics. The weight loss rates of PUR foam and polyester plastic PCL were 33.88% and 83.44% respectively within 2 days, and the degradation rates of agricultural degradable mulch PBAT and PU plastic oligomer PBA-PU were 66.70% and 64.41% respectively within 4 days. The results showed that cutinase BaCut1 had good polyester plastic degradation ability.

Claims

1. A keratinase gene, characterized in that... The full-length nucleotide sequence of this gene is: SEQ ID NO.

1.

2. The keratinase BaCut1 encoded by the keratinase gene of claim 1, characterized in that... The amino acid sequence is: SEQ ID NO.

2.

3. A recombinant plasmid containing the keratinase gene of claim 1.

4. The recombinant plasmid according to claim 3, characterized in that... The recombinant plasmid is obtained by cloning the keratinase gene of claim 1 into plasmid pET29a or pEFαA.

5. Recombinant microorganisms containing the keratinase gene of claim 1 or the recombinant plasmid of claim 3 or 4.

6. The recombinant microorganism according to claim 5, characterized in that... Escherichia coli or yeast are used as host bacteria.

7. The genetic engineering application of the keratinase gene of claim 1, the recombinant plasmid of claim 3 or 4, and the recombinant microorganism of claim 5 in the biodegradation of polyester plastics.

8. The application according to claim 7, characterized in that... The polyester plastic is selected from agricultural mulch film PBAT and biodegradable plastic PCL.

9. The genetic engineering application of the keratinase gene of claim 1, the recombinant plasmid of claim 3 or 4, and the recombinant microorganism of claim 5 in the biodegradation of PUR foam.

10. The application of the keratinase BaCut1 according to claim 2 in the biodegradation of polyester plastics.

11. The application according to claim 10, characterized in that... The polyester plastic is selected from agricultural mulch film PBAT and biodegradable plastic PCL.

12. The application of the keratinase BaCut1 according to claim 2 in the biodegradation of PUR foam.

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