Cutinase mutant and application thereof to efficient degradation of pet

Abstract

The application discloses a cutinase mutant and application thereof to efficient degradation of PET. The amino acid sequence of the cutinase mutant is shown in SEQ ID NO. 2, and is obtained by replacing phenylalanine at the 239th amino acid in the amino acid sequence of a wild-type cutinase shown in SEQ ID NO. 1 with alanine. The cutinase mutant is expressed in E. coli, and compared with the wild-type enzyme, the activity of the mutant enzyme is increased in stability, the catalytic efficiency is improved, and the yield of a PET plastic (polyethylene terephthalate, PET) degradation product is increased by 42.6 times. The improved cutinase mutant improves the PET degradation efficiency, reduces the production cost, and is more suitable for industrial application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, and particularly relates to a cutinase mutant and application thereof to efficient degradation of PET. BACKGROUND

[0002] The rapid development of the plastic industry has changed people's traditional production and living ways, and has brought great convenience to people's life, but a large amount of plastic waste produced has been accumulated in the natural environment, causing a serious burden to the global ecological environment. A large amount of plastic waste enters the marine ecological system every year, causing a serious survival threat to birds, fish and other organisms in the sea. Polyethylene glycol terephthalate (PET) is synthesized from petroleum-derived terephthalic acid and ethylene glycol, has the characteristics of long hydrocarbon chain, high molecular weight, low gas permeability and difficult degradation, etc. Due to the advantages of low production cost, good durability and easy use, PET has become the most used polyester material, and more than half of the synthetic fibers and plastic bottles in the world are made of PET. Since the main chain of PET molecules contains aromatic compounds and is difficult to be naturally degraded, the development of green degradation treatment technology for PET plastics has important significance for global ecological environment protection.

[0003] Cutinase belongs to the alpha / beta fold hydrolase superfamily, has a catalytic triad composed of serine, histidine and aspartic acid residues, and is an important enzyme for effectively degrading PET. Under the action of the enzyme, PET is degraded into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA), which can be effectively recycled and reused. The glass transition temperature of PET plastic is 76℃, and the cutinase stable at high temperature is an important group for degrading PET. Protein engineering of cutinase can significantly improve the PET degradation activity, thermal stability and environmental adaptation potential of the wild-type enzyme. The cutinase ScCut from Nocardioides sp. SCSIO 66511 has an optimal enzyme reaction temperature of 70℃, but the activity is relatively low. Therefore, the mutant with high activity is constructed by structure analysis in the present patent, and the mutant shows good industrial application potential in the degradation of waste PET plastics in the environment. SUMMARY

[0004] The first object of the present application is to provide a cutinase mutant, the amino acid sequence of which is shown in SEQ ID NO. 2. The cutinase mutant is obtained by replacing phenylalanine at position 239 with alanine based on the wild-type cutinase shown in SEQ ID NO. 1.

[0005] A second object of the present application is to provide a coding gene encoding the cutinase mutant described above. Preferably, the nucleotide sequence thereof is shown in SEQ ID NO. 4.

[0006] A third object of the present application is to provide a recombinant vector containing the coding gene described above.

[0007] A fourth object of the present application is to provide a recombinant engineering bacterium containing the recombinant vector described above.

[0008] Preferably, the recombinant engineering bacterium is Escherichia coli.

[0009] Preferably, the Escherichia coli is BL21(DE3).

[0010] A fifth object of the present application is to provide the use of the cutinase mutant described above, the recombinant vector described above or the recombinant engineering bacterium described above in PET degradation.

[0011] A sixth object of the present application is to provide a method for degrading PET, which is to react the cutinase mutant described above with PET.

[0012] Preferably, the concentration of the cutinase mutant is 1 mg / mL.

[0013] The present application determines the binding pocket of cutinase and substrate through molecular docking simulation, finds the key amino acid residues (position 239) affecting activity, improves the catalytic efficiency of PET through site-directed mutation, and increases the release amount of degradation products. Compared with the wild-type strain, the cutinase mutant catalyzes the product by 42.6 times, and the degradation activity exceeds one of the best degradation activity enzymes ICCG in the world, showing better industrial application potential in reducing production cost and improving production efficiency.

[0014] Nocardioides sp. SCSIO 66511 is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 26044, the preservation address being No. 1, Xibei Road, 3, Haidian, Beijing, China, the postal code being 100101, and the preservation date being December 4, 2022. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a schematic diagram of the docking of cutinase ScCut and molecules MHET and BHET; (A, docking diagram of molecule MHET; B, docking diagram of molecule BHET).

[0016] Figure 2 is a schematic diagram of the comparison of the structure of the wild-type enzyme and the mutant calculated by simulation; (green, wild-type enzyme structure; blue, mutant structure).

[0017] Figure 3 is the liquid chromatography peak of PET degradation product (wild type enzyme ScCut; mutant enzyme ScCut-Phe239Ala; control enzyme ICCG, Control is the baseline of HPLC detection).

[0018] Figure 4 is the aromatic compound contrast of degradation product (wild type enzyme ScCut; mutant enzyme ScCut-Phe239Ala; control enzyme ICCG). DETAILED DESCRIPTION

[0019] In the following examples, the specific molecular biology experimental methods are not specifically described, and are carried out according to the specific methods listed in the book of Molecular Cloning Experiments Guide (third edition) J. Sambrook, or according to the kit and product instructions.

[0020] The following definitions are used in the present application:

[0021] 1. Nomenclature of amino acid and DNA nucleotide sequences

[0022] The recognized IUPAC nomenclature of amino acid residues is used in the form of three-letter code. The recognized IUPAC nomenclature of DNA nucleotide sequences is used.

[0023] 2. Identification of cutinase mutants, the amino acid of “original amino acid position replaced by amino acid” is used to represent the mutated amino acid in ScCut mutant. For example, Phe239Ala, which means the amino acid at position 239 is replaced by Ala from the wild type Phe, and the position number corresponds to the amino acid sequence number of wild type ScCut in SEQ ID No. 1.

[0024] The present application will be further explained in conjunction with the accompanying drawings and examples.

[0025] Example 1: Wild type cutinase and molecular docking of degradation products MHET and BHET

[0026] Taking cutinase ScCut gene as a template, protein structure prediction is carried out in Alpha fold 2, and docking is carried out by using AutoDock4.2 software, and according to the binding mode, it is found that there is a structure similar to “cover” on the upper part of the substrate binding pocket ( Figure 1 ), which hinders the entry of the reaction substrate. Therefore, in order to improve the activity, the mutation of the “cover” structure is selected, and after replacing the phenylalanine at position 239 with alanine, the substrate binding pocket is obviously larger ( Figure 2 ).

[0027] Example 2: Obtaining site-directed mutant strains

[0028] The gene encoding ScCut enzyme (nucleotides 91-951 of SEQ ID NO. 3, 1-90 is the signal peptide sequence) was amplified using the genome of marine Nocardioides sp. SCSIO 66511 as a template and SC-F1 (5'-gga gatatacatatgGATGCACCGTGCGCGCCGCT-3') and SC-R1 (5'-ggtgctcgagGCGTGCCGCGTCGCGCAGGC-3') as primers. The PCR reaction system is shown in Table 1, and the PCR reaction procedure was as follows: pre-denaturation at 95 °C for 10 min; 32 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min; extension at 72 °C for 10 min; and preservation at 4 °C. The amplified PCR product was purified using a PCR purification kit. The purified product was subjected to homologous recombination with linearized vector pET-22b(+) (doubly digested with Nde I and Xho I) at 50 °C for 5 min. 5 μL of the recombination product was added to 100 μL of competent cells, which were then subjected to ice bath for 30 min, heat shock at 42 °C for 30 s, ice bath for 2 min, and centrifugation. Then, 500 μL of LB medium (without antibiotics) was added to the centrifugal tube, which was then incubated at 37 °C and 120 r / min for 1 h. The E. coli cells were then spread on an LB culture dish containing ampicillin (100 μg / mL) and incubated at 37 °C for 12 h. Single colonies were picked and subjected to sequencing verification. The E. coli DH5a (pET-22b-ScCut) verified to be successful was expanded and cultured, and the plasmid was extracted for standby use.

[0029] Table 1 PCR reaction system

[0030]

[0031] The nucleotide sequence was mutated from SEQ ID NO. 3 to SEQ ID NO. 4 based on the method of one-step site-directed mutagenesis, i.e. TTC at 715-717 of SEQ ID NO. 3 was mutated to GCA in SEQ ID NO. 4, i.e. the amino acid was replaced from wild-type Phe to Ala, to obtain the mutant. The circular plasmid pET-22b-ScCut was used as a template, and primers 239M-F1 (5'-GACACGTCGATCGCACAGCAGGCCCTGAAGATG-3') and 239M-R1 (5'-TGCGATCGACGTGTCTGCGAGTGCGGACTTGCC-3') were used for amplification; the PCR reaction system is shown in Table 1, and the PCR reaction procedure was as follows: pre-denaturation at 95°C for 10 min; 32 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 4 min; extension at 72°C for 10 min; preservation at 4°C. The amplified PCR product was purified using a PCR purification kit to obtain a linearized plasmid; 5 μL of the purified product (linearized plasmid) was added to 100 μL of E. coli XL1-Blue competent cells, which were subjected to ice bath for 30 min, then heat shock at 42°C for 30 s, ice bath reaction for 2 min, and then 500 μL of LB medium (without antibiotics) was added to the centrifugal tube, which was cultured at 37°C and 120 r / min for 1 h; then the E. coli cells were spread on an LB culture dish containing ampicillin (100 μg / mL), which was cultured at 37°C for 12 h; single colonies were picked and subjected to sequencing verification (amplification primers were SC-F1 / R1), and it was confirmed that the nucleotide at position 715-717 was GCA. The successfully verified E. coli XL1-Blue (pET-22b-ScCut-Phe239Ala) was cultured on a large scale, and the plasmid was extracted for standby use. The extracted plasmid pET-22b-ScCut-Phe239Ala was transformed into E. coli BL21 (DE3) competent cells, which were cultured at 37°C and 120 r / min for 1 h; then the E. coli cells were spread on an LB culture dish containing ampicillin (100 μg / mL), which was cultured at 37°C for 12 h; single colonies were picked and subjected to sequencing verification, and the successfully verified one was the genetically engineered bacteria BL21 (DE3) / ScCut-Phe239Ala containing cutinase.

[0032] In the same way, pET-22b-ScCut was transformed into E. coli BL21 (DE3) to obtain the genetically engineered bacteria BL21 (DE3) / ScCut containing wild-type cutinase.

[0033] Example 3: Preparation of novel cutinase ScCut-Phe239Ala protein

[0034] The genetically engineered bacteria BL21(DE3) / ScCut-Phe239Ala(or genetically engineered bacteria BL21(DE3) / ScCut) were cultured in a 500 mL conical flask containing 100 mL of LB liquid medium at 37°C, 180 rpm / min for 4 h, inoculated into LB liquid medium containing ampicillin(100 μg / mL) at a volume ratio of 2%, and cultured at 37°C, 180 rpm / min until the OD600 was 0.6-0.8; then 0.1 mM IPTG was added, and the culture was incubated at 16°C for 20 h. The bacterial cells were collected by centrifugation at 8000 r / min for 10 min at 4°C, washed with Tris-HCl buffer(50 mM, pH 8.0) for 3 times, resuspended and ultrasonically broken, then centrifuged at 10000 r / min at 4°C to remove the precipitate, and the obtained supernatant was the crude enzyme solution. The crude enzyme solution was subjected to protein purification using a Ni 2+ affinity chromatography column, eluted with an imidazole gradient, collected, then replaced with 50 mM Tris-HCl buffer(pH 8.0) using a 10 kDa ultrafiltration tube, and concentrated. The purity of the protein was tested by SDS-PAGE, and the concentration of the concentrated protein was detected by a microprotein detector. Thus, the cutinase ScCut and the cutinase ScCut-Phe239Ala were obtained.

[0035] Example 4: Effect of site-directed mutant enzyme of cutinase on PET plastic degradation efficiency

[0036] The degradation experiment of the cutinase ScCut and the cutinase ScCut-Phe239Ala on PET plastic film was carried out according to the following steps:

[0037] The PET film was cut into 1 cm 2 size, then sterilized by soaking in 75% ethanol for 1 h, and the plastic film was taken out and placed in a sterile culture dish, and the ethanol on the surface of the plastic was volatilized in a sterile operation table. In a 10 mL glass bottle, 2 mL of 20 mM Tris-HCl(pH 8.5) was added, then the purified cutinase was added to a final concentration of 1 mg / mL, and 3 pieces of sterile treated PET plastic film were placed in the bottle; the control group was added with 2 mL of 20 mM Tris-HCl(pH 8.5), and 3 pieces of sterile treated PET plastic film were placed in the bottle. Each group was set in triplicate, and the conical flask was placed in a constant temperature shaker at 70°C, 180 r / min for 48 h, and the PET degradation products were detected. ICCG enzyme was used as a control.

[0038] PET degradation product analysis process: take 1 mL of the degradation solution, centrifuge at 8000 rpm / min for 10 min, filter the supernatant using a 0.22 μm water filter membrane, and reserve. Use Agilent 1260 high performance liquid chromatography to detect the composition of TPA, MHET and BHET. Chromatographic column; Zorbax SB-C18 ODS (4.6*150mm, 5 μm), detection wavelength 240 nm. Mobile phase: A is deionized water containing 0.1% (v / v) formic acid; B is methanol containing 0.1% (v / v) formic acid. Gradient elution condition: 0-5 min 10% B; 5-20 min 10%-100% B solution; flow rate 1.0 mL / min, column temperature 30°C.

[0039] It is determined that the keratinase PET degradation activity after mutation is significantly improved. At 70°C, the mutant enzyme can degrade PET and release 407.5 μM of aromatic compounds after 48 h of reaction, which is 42.6 times the product released by the wild enzyme ( Figure 4 ), indicating that the mutation effect is obvious. The degradation product detected by liquid phase is mainly TPA, and the degradation effect is better than that of ICCG enzyme which has been studied a lot ( Figure 3 ).

[0040] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are within the protection scope of the present application. SEQ ID NO. 1 (wild-type enzyme ScCut amino acid sequence)

[0041] MKVRALLASAAMVAGGSMLAVGSTTAPASADAPCAPLQVVGVPGTGYELIFDGQGMPNGLDLLKPGVLIKDVAAKLQPERDAGRVTYQQVPYPADIGI T MSYRKSVRVGTAATKTYIKAKSIQCPGSRFALIGYSQGARVAGNVLHSIGKGNGPIAPDKLAVGGLWSDPGRSTSDQLIGPGVPGVGIDRLRKGGFGAVNGRTFSVCAPGDIVCSTDDTTLLRPLVRKFGKSALADTSIFQQALKMLRRNGFDLQKWSREFGEQDPMSLLPKAFKTGFEIDRYVREGSHGHYLVGNTISVDGQSSIDWVTDRLRDAARSEQ ID NO. 2 (mutant enzyme ScCut-Phe239Ala amino acid sequence)

[0042] MKVRALLASAAMVAGGSMLAVGSTTAPASADAPCAPLQVVGVPGTGYELIFDGQGMPNGLDLLKPGVLIKDVAAKLQPERDAGRVTYQQVPYPADIGITMSYRKSVRVGTAATKTYIKAKSIQCPGSRFALIGYSQGARVAGNVLHSIGKGNGPIAPDKLAVGGLWSDPGRSTSDQLIGPGVPGVGIDRLRKGGFGAVNGRTFSVCAPGDIVCSTDDTTLLRPLVRKFGKSALADTSIAQQALKMLRRNGFDLQKWSREFGEQDPMSLLPKAFKTGFEIDRYVREGSHGHYLVGNTISVDGQSSIDWVTDRLRDAARSEQ ID NO. 3 (nucleotide sequence encoding wild-type ScCut enzyme)

[0043] ATGAAGGTACGAGCACTACTGGCCTCCGCAGCAATGGTCGCGGGTGGCTCGATGTTGGCTGTCGGGTCCACTACTGCGCCGGCAAGCGCA GATGCACCGTGCGCGCCGCTGCAGGTCGTCGGCGTTCCAGGCACCGGGTACGAGCTCATCTTCGACGGGCAAGGTATGCCGAACGGCCTCGATCTGCTCAAGCCAGGCGTGTTGATCAAGGATGTGGCCGCCAAGTTGCAGCCTGAGCGCGACGCAGGCAGGGTCACCTACCAGCAGGTCCCCTACCCCGCGGACATCGGCATCACCATGTCGTACCGCAAGTCGGTCCGGGTCGGAACGGCCGCTACGAAGACCTACATCAAGGCAAAGTCGATCCAGTGCCCGGGCAGCCGGTTCGCGCTCATCGGGTACTCGCAGGGTGCGAGGGTCGCCGGAAACGTGCTGCATTCCATCGGCAAGGGCAACGGGCCGATCGCGCCCGATAAGCTCGCGGTCGGCGGCTTGTGGTCCGATCCCGGCCGATCGACGTCCGACCAGCTGATCGGACCGGGAGTACCGGGTGTTGGCATCGACCGGCTACGCAAGGGAGGCTTCGGCGCGGTCAACGGCCGTACCTTCTCGGTCTGCGCTCCCGGCGACATCGTCTGCTCGACGGACGATACGACGCTGCTGCGTCCGCTCGTGCGCAAGTTCGGCAAGTCCGCACTCGCAGACACGTCGATCTTCCAGCAGGCCCTGAAGATGCTCCGCCGCAACGGATTCGACCTACAGAAGTGGTCGCGCGAGTTCGGTGAGCAGGATCCGATGTCGCTGCTGCCGAAGGCGTTCAAGACCGGGTTCGAGATCGACCGGTACGTCCGCGAGGGCAGCCACGGCCATTACCTCGTCGGCAACACCATCAGTGTCGACGGCCAGTCGTCGATCGACTGGGTGACGGATC GCCTGCGCGACGCGGCACGC SEQ ID NO.4 (Nucleotide sequence encoding mutant enzyme ScCut-Phe239Ala)

[0044] ATGAAGGTACGAGCACTACTGGCCTCCGCAGCAATGGTCGCGGGTGGCTCGATGTTGGCTGTCGGGTCCACTACTGCGCCGGCAAGCGCA GATGCACCGTGCGCGCCGCTGCAGGTCGTCGGCGTTCCAGGCACCGGGTACGAGCTCATCTTCGACGGGCAAGGTATGCCGAACGGCCTCGATCTGCTCAAGCCAGGCGTGTTGATCAAGGATGTGGCCGCCAAGTTGCAGCCTGAGCGCGACGCAGGCAGGGTCACCTACCAGCAGGTCCCCTACCCCGCGGACATCGGCATCACCATGTCGTACCGCAAGTCGGTCCGGGTCGGAACGGCCGCTACGAAGACCTACATCAAGGCAAAGTCGATCCAGTGCCCGGGCAGCCGGTTCGCGCTCATCGGGTACTCGCAGGGTGCGAGGGTCGCCGGAAACGTGCTGCATTCCATCGGCAAGGGCAACGGGCCGATCGCGCCCGATAAGCTCGCGGTCGGCGGCTTGTGGTCCGATCCCGGCCGATCGACGTCCGACCAGCTGATCGGACCGGGAGTACCGGGTGTTGGCATCGACCGGCTACGCAAGGGAGGCTTCGGCGCGGTCAACGGCCGTACCTTCTCGGTCTGCGCTCCCGGCGACATCGTCTGCTCGACGGACGATACGACGCTGCTGCGTCCGCTCGTGCGCAAGTTCGGCAAGTCCGCACTCGC AGACACGTCGATCGCACAGCAGGCCCTGAAGATG CTCCGCCGCAACGGATTCGACCTACAGAAGTGGTCGCGCGAGTTCGGTGAGCAGGATCCGATGTCGCTGCTGCCGAAGGCGTTCAAGACCGGGTTCGAGATCGACCGGTACGTCCGCGAGGGCAGCCACGGCCATTACCTCGTCGGCAACACCATCAGTGTCGACGGCCAGTCGTCGATCGACTGGGTGACGGATC GCCTGCGCGACGCGGCACGC 。

Claims

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

2.

2. A coding gene encoding the cutinase mutant of claim 1.

3. The genetic code according to claim 2, wherein, The nucleotide sequence is shown as SEQ ID NO.

4.

4. A recombinant vector containing the coding gene of claim 2.

5. A recombinant engineering bacteria containing the recombinant vector of claim 4.

6. The recombineering bacteria of claim 5, wherein, The recombinant engineering bacteria is Escherichia coli.

7. The recombineering bacteria of claim 6, wherein, The Escherichia coli is BL21 (DE3).

8. The cutinase mutant of claim 1, the recombinant vector of claim 4 or the recombinant engineering bacteria of claim 5 in the application of PET degradation.

9. A method of degrading PET, characterized by, The cutinase mutant of claim 1 is used to react with PET.

10. The method of claim 9, wherein, The concentration of the cutinase mutant is 1 mg / mL.

Images