A salt-tolerant cutinase mutant of hydrolyzed polyethylene terephthalate and applications thereof

Abstract

The application discloses a salt-tolerant cutinase mutant of hydrolyzed polyethylene terephthalate and application thereof. Streptomyces alkaliphilus PET cutinase Sa Cut27 is obtained through mutation, and the amino acid sequences are shown in SEQ ID NO. 3-15 in sequence. PET cutinase Sa Cut27 can exhibit PET degradation activity in a high-salt environment, and 4.17 mM of degradation products can be obtained under the action of 2 M NaCl at 50 DEG C for 48 h, which is better than Is PETase. Compared with the original PET degradation enzyme, the heat stability and / or specific activity of the obtained mutant are significantly improved. The enzyme has wide application prospects in the fields of marine plastic recycling, high-salt wastewater treatment and high-ion-strength reaction systems.

Description

Technical Field

[0001] This invention belongs to the field of environmental science, specifically relating to a keratinase that efficiently hydrolyzes PET plastic under high-salt conditions, its mutation, and its application. Background Technology

[0002] PET is one of the most produced polyester plastics, widely used in beverage bottles, fibers, films, and other fields. Due to its high crystallinity and hydrophobicity, PET degrades extremely slowly. Several PET hydrolytic enzymes (such as...) have been discovered. Is PETase, LCC, Tf (Examples of enzymes include Cut2 and HiC), but these enzymes exhibit significantly reduced activity in high-salt or high-ionic-strength systems, making them unsuitable for marine plastic recycling and saline industrial reactions. Therefore, it is necessary to develop a PET hydrolase that retains high activity and stability under high-salt conditions to expand its industrial applications and improve depolymerization efficiency. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a keratinase capable of hydrolyzing PET in a high-salt environment and its applications.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A salt-resistant keratinase for hydrolyzing PET, said enzyme being derived from... Streptomyces alkaliphilus Named Sa Cut27, whose amino acid sequence is shown in SEQ ID NO.1.

[0005] The keratinase Sa The nucleotide sequence of Cut27 is shown in SEQ ID NO. 2.

[0006] A keratinase Sa The genetically engineered bacteria of Cut27, using the DH5α plasmid as a vector and Escherichia coli BL21(DE3) as a host, encodes... Sa The Cut27 gene was obtained.

[0007] Keratinase Sa Cut27 exhibits high activity at pH 7-9, with an optimal pH of 8.0; Keratinase Sa Cut27 is active at 20-80°C, with an optimal temperature of up to 50°C, and retains significant activity at 60-70°C. At different NaCl concentrations, Sa Cut27 exhibits significantly different salt response characteristics. SaCut27 exhibits stable activity under 0–1 M conditions, reaching its highest activity at 2 M NaCl, demonstrating a significant “salt-activated” effect. Even in high-salt environments of 3–5 M, its activity can still be maintained at 25–55%, exhibiting remarkable salt tolerance.

[0008] at the same time Sa Cut27 is resistant to metal ions and organic solvents. 2+ Mg 2+ It has a certain activating effect on enzyme activity, and the metal ion Mn 2+ right Sa Cut27 has little effect; dimethyl sulfoxide has the most significant effect on improving enzyme activity, while acetonitrile, isopropanol, and acetone can maintain or slightly improve enzyme activity.

[0009] A keratinase mutant resistant to salt-hydrolyzed PET, wherein the amino acid sequence of the mutant is as shown in SEQ ID No. 1. Sa Cut27 was obtained by mutating S to G at position 121 of its amino acid sequence (amino acid sequence shown in SEQ ID NO.3). Alternatively, it can be obtained by mutating S to D at position 121 (amino acid sequence as shown in SEQ ID NO.4). Alternatively, it can be obtained by mutating S to E at position 123 (amino acid sequence as shown in SEQ ID NO.5). Alternatively, it can be obtained by mutating Q at position 150 to G (amino acid sequence as shown in SEQ ID NO.6). Alternatively, it can be obtained by mutating D to C at position 260 (amino acid sequence as shown in SEQ ID NO.7); Alternatively, it can be obtained by mutating S to Q at position 122 (amino acid sequence as shown in SEQ ID NO.8); Alternatively, it can be obtained by mutating L at position 148 to F (amino acid sequence shown in SEQ ID NO.9). Alternatively, it can be obtained by mutating I at position 197 to A (amino acid sequence as shown in SEQ ID NO.10). Alternatively, it can be obtained by mutating P at position 210 to G (amino acid sequence shown in SEQ ID NO.11). Alternatively, it can be obtained by mutating H at position 240 to Y (the amino acid sequence is shown in SEQ ID NO.12).

[0010] Preferably, the present invention provides a salt-tolerant keratinase mutant of PET, wherein the amino acid sequence of the mutant is derived from the wild-type PET depolymerase. SaThe amino acid sequence of Cut27 was obtained by mutating S to G at position 121, L to F at position 148, and H to Y at position 240. Its amino acid sequence is shown in SEQ ID NO.13. Alternatively, the amino acid sequence obtained by mutating S at position 121 to G, D at position 260 to C, and H at position 240 to Y is shown in SEQ ID NO.14. Alternatively, the amino acid sequence obtained by mutating L at position 148 to F, D at position 260 to C, and H at position 240 to Y is shown in SEQ ID NO.15.

[0011] Gene encoding a keratinase mutant that is tolerant to salt hydrolysis of PET.

[0012] The gene encoding the PET depolymerase mutant S121G / L148F / H240Y has the nucleotide sequence shown in SEQ ID NO.16.

[0013] A nucleic acid molecule encoding a keratinase mutant of the salt-tolerant hydrolyzed PET.

[0014] A recombinant expression vector containing the aforementioned nucleic acid molecule.

[0015] A host cell containing the recombinant expression vector, or having the nucleic acid molecule integrated into its chromosome.

[0016] A method for preparing the salt-tolerant hydrolyzed PET keratinase mutant involves culturing the host cells and separating and purifying the expression product to obtain the salt-tolerant hydrolyzed PET keratinase mutant.

[0017] The application of the salt-tolerant hydrolyzed PET keratinase mutant, the nucleic acid molecule, the recombinant expression vector, or the host cell in the depolymerization of PET plastic.

[0018] The application is carried out in a salt-containing reaction system with a salt concentration of 1–5 M.

[0019] The host cell is *Escherichia coli*, which is used to produce keratinase. Sa The Cut27 method includes the following steps: Inoculate the seed culture at a volume fraction of 1% (v / v) into 5 mL of LB liquid medium containing kanamycin, and incubate at 37°C for approximately 12 h to obtain primary bacterial culture; then transfer the 1% (v / v) inoculation to 100 mL of LB medium containing kanamycin, and continue incubation until OD (dose retardation). 600 After adjusting the concentration to 0.6–0.8, IPTG was added for low-temperature induction for 24 h; subsequently, the bacterial culture was collected, centrifuged to obtain bacterial cells, which were then broken up and purified to obtain the final product.Sa Cut27 mutant.

[0020] The above-mentioned keratinase Sa Application of Cut27 and its mutants in the degradation of PET plastics.

[0021] PET is degraded under high salt or high temperature conditions to produce mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA).

[0022] The plastic is pure polyester, or PET plastic, or a mixture of polyester with cotton, spandex, nylon, and acrylic in any proportion. The keratinase... Sa The reaction of Cut27 and its mutants with PET resulted in a solid content of 2-5 g / L and an enzyme-to-solid ratio of 2‰ (m / m).

[0023] The salt-tolerant hydrolyzed PET keratinase depolymerizes PET under conditions of 20–60°C and pH 7.0–10.0.

[0024] Preferably, the keratinase Sa The depolymerization temperature of Cut27 is 45-55℃, and the depolymerization pH is 7.5-8.5.

[0025] Beneficial effects:

[0026] This invention provides a novel salt-resistant polyethylene terephthalate hydrolase-keratinase. Sa Cut27 possesses the ability to efficiently catalyze the hydrolysis of ester bonds in plastics, enabling the efficient depolymerization of PET plastics. Further modifications to the enzyme based on its activity and thermal stability yielded mutants with significantly enhanced thermal stability and / or specific activity, showing broad application prospects in marine plastic recycling, high-salinity wastewater treatment, and high-ionic-strength reaction systems. Attached Figure Description

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the objectives, technical solutions and other advantages of the present invention will become clearer.

[0028] Figure 1 To express Sa Plasmid map (A) and SDS-PAGE electrophoresis image (B) of Cut27: M, protein marker; T, whole cell; P, lysate after disruption; SN, supernatant after disruption; E, purified. Sa Cut27 ； Figure 2 For amino acid sequence alignment (A) and phylogenetic tree analysis (B); Figure 3 Keratinase SaEnzymatic properties of Cut27; where: A: temperature; B: pH; C: salt concentration; D: metal ions; E: organic solvent; Figure 4 Keratinase Sa Evaluation of Cut27 PET depolymerization by high performance liquid chromatography (A) and electron microscopy characterization (B); Figure 5 Keratinase Sa Evaluation of Cut27 mutants by high-performance liquid chromatography. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the plastics used are pure polyester, PET plastic, or a mixture of polyester with cotton, spandex, nylon, and acrylic in any proportion.

[0030] This invention screened and obtained an enzyme, which was named Sa Cut27. The functional properties and catalytic activity of this enzyme were tested and verified through experiments.

[0031] In this invention, keratinase Sa Cut27 protein expression strain BL21 / DH5α- Sa Cut27 was constructed by Nanjing GenScript, and the intracellular enzyme was obtained from the E. coli expression system. The culture medium used was LB medium, and the antibiotic used was kanamycin.

[0032] Inoculate the culture at a 1% (v / v) inoculum into LB liquid medium containing 0.05 mg / mL kanamycin resistance and incubate at 37°C for 12 h. Then, add the bacterial culture to the LB liquid medium at a 1% (v / v) inoculum and incubate at 37°C for 2.5 h to reach the logarithmic growth phase. Add IPTG to a final concentration of 0.1 mM and incubate at 18°C ​​for 24 h. After centrifugation, obtain the bacterial cells. The basic LB culture formula is: 10 g / L peptone, 10 g / L glucose, and 5 g / L yeast extract.

[0033] The centrifuged bacterial cells were resuspended in 50 mL of PBS and sonicated in an ice bath. The sonication program was 150 W for 4 seconds followed by a 4-second pause, for a total sonication time of 15 min. The supernatant was collected by centrifugation at 8000 rpm for 10 min at 4°C. The supernatant was incubated with 3 mL of Ni-NTA resin at 4°C for 30 min to allow the target protein to bind to the resin. The cells were then washed with 20-500 mM imidazole, and the target protein was collected. The eluent was concentrated to 5 mL using a 10 kDa ultrafiltration tube. Sa Cut27 keratinase.

[0034] In this invention, the enzyme activity assay method is as follows: using p-NPO as a substrate, the reaction is carried out at 37°C for 5 min. The amount of enzyme required to produce p-nitrophenol per unit time is defined as one enzyme activity unit (U). The protease activity formula is: Protease activity = A / (B*C), where A is the amount of p-nitrophenol produced (μmol), B is the reaction time (min), and C is the amount of enzyme added to the reaction (mL).

[0035] The phosphate-buffered saline (PBS) used in the embodiments of this invention is 50 mM PBS with a pH of 7.5-8.5. In the following embodiments, various types of PET were depolymerized, and the products (TPA, MHET) were evaluated by liquid chromatography. Sa The degradation effect of Cut27 on PET.

[0036] The following example illustrates the liquid chromatography (LC) detection method for the main depolymerization products. The laboratory was equipped with an Agilent 1260 Infinity II high-performance liquid chromatography system (Agilent Technologies, Inc., USA). Specific detection conditions were as follows: LC column: Agilent 5 HC-C18 (2) 150 × 4.6 mm, 5 μm; UV detection wavelength: 240 nm; column oven temperature: 30℃; mobile phase flow rate: 0.8 mL / min; autosampler injection volume: 10 μL; mobile phase consisted of 18% acetonitrile, 1% formic acid, and 81% water (pH 2.5). The concentrations of TPA and MHET were calculated based on standard samples.

[0037] Example 1: Keratinase Sa Plasmid mapping and SDS-PAGE electrophoresis of Cut27 pass Sa The entire genome was synthesized using the Cut27 amino acid sequence, and codon optimization was performed for expression in *E. coli*. The synthesized sequence is shown in SEQ ID No. 2. The diagram is as follows: Figure 1 As shown in (A), the expression strain PET-29a(+)- was obtained. Sa Cut 27 The protein expression fusion protein with a C-terminal Trx-His6 tag was purified for subsequent experiments. Whole cells were harvested, lysed, and the supernatant and purified protein were analyzed by SDS-PAGE. The results are shown below. Figure 1 As shown in (B), the protein size is 35.3 kDa.

[0038] Example 2: Keratinase Sa Amino acid sequence analysis and phylogenetic analysis of Cut27 like Figure 2 As shown in (A), in order to determine SaThe novelty of the Cut27 amino acid sequence was assessed by searching for its homologous proteins in GenBank using online BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), selecting those that were similar to the Cut27 amino acid sequence. Sa Sequences with high Cut27 similarity participated in phylogenetic tree construction. Amino acid sequence analysis revealed that ( Figure 2 (B) Sa Cut27 possesses the typical triplet catalytic active site S186–D232–H264 of the α / β hydrolase superfamily proteins, as well as the characteristic sequence tag (G–X–S–X–G) of this family of proteins. Simultaneously, this enzyme interacts with… Streptomyces calidiresistens The keratinase showed the highest sequence similarity, reaching 94.6%, thus identifying it as a keratinase belonging to the α / β hydrolase superfamily. The phylogenetic tree shows... Sa Cut27 and previously reported PET hydrolases Is The similarity to PETase, LCC, and PET2 was 47.89%, 53.37%, and 43.94%, respectively, indicating that this protein (SEQ ID No. 2) is a novel plastic-degrading enzyme.

[0039] Example 3: Keratinase Sa Cut27 Enzymatic Properties Assay Keratinase Sa The enzymatic properties of Cut27 include the optimal temperature and pH for enzyme activity, the optimal salt concentration, and the effects of metal ions and chemical reagents. Sa Effect of Cut27 enzyme activity.

[0040] Determination of the optimal reaction pH of the enzyme: The reaction system was placed in the activity assay system of 50 mM citrate buffer (pH 4.0-6.0), 50 mM PBS buffer (pH 6.0-8.0) and 50 mM glycine-NaOH buffer (pH 8.0-10.0), respectively. After reacting at 55℃ for 10 min, the enzyme activity was measured to determine the optimal reaction pH of the enzyme. The reaction system was placed in water baths at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃ and 80℃ for 10 min to determine the optimal reaction temperature of the enzyme. Keratinase Sa Cut27 was added to a solution containing 1 mM of different metal ions (Ca). 2+ Mn 2+ Mg 2+ Al 3+ Cu 2+ Zn 2+In an activity assay system containing 10% (v / v) organic solvents (acetone, isopropanol, methanol, ethanol, DMSO, N,N-dimethylformamide, dithiothreitol), the reaction was carried out at 37°C for 5 min. The enzyme activity of the blank group was set as 100%, and the relative enzyme activity after the addition of metal ions and chemical reagents was calculated.

[0041] Depend on Figure 3 As can be seen from Figure B, Sa Cut27 and Is PETase is almost inactive under acidic conditions (pH 3–6), indicating that both PETase and PETase prefer neutral or weakly alkaline environments. Sa Cut27 exhibits high activity at pH 7-9, with an optimal pH of 8.0; Depend on Figure 3 As shown in Figure A, the control group Is PETase and keratinase Sa Cut27 is active at temperatures ranging from 20 to 80°C, compared to... Is PETase, keratinase Sa Cut27 has an optimal temperature of up to 50°C and maintains significant activity at 60–70°C, exhibiting significantly superior performance compared to other treatments. Is PETase.

[0042] Depend on Figure 3 As shown in Figure C, at different NaCl concentrations, Sa Cut27 exhibits significantly different salt response characteristics. Sa Cut27 exhibits stable activity at 0–1 M, reaching its peak activity at 2 M NaCl, demonstrating a significant "salt activation" effect; even in high-salt environments of 3–5 M, its activity remains at 25–55%. In contrast, Is PETase is highly sensitive to salt; its activity decreases continuously with increasing salt concentration and it is completely inactivated at 4–5 M. Overall, Sa Cut27 exhibits significant salt activation and salt tolerance properties, while Is PETase is rapidly deactivated under high-salt conditions.

[0043] Depend on Figure 3 As shown in Figure D, metal ions affect keratinase. Sa The enzyme activity of Cut27 is affected to some extent, including the metal ion Ca. 2+ Mg 2+ It has a certain activating effect on enzyme activity, with relative enzyme activities of 123.3% and 138.9%, respectively; metal ion Mn 2+ right Sa Cut27 has little impact; Cu 2+ Zn 2+ And Al2+ It has an inhibitory effect on enzyme activity.

[0044] Depend on Figure 3 As shown in Figure E, dimethyl sulfoxide (DTT) significantly enhances enzyme activity, exhibiting the highest relative activity. Acetonitrile, isopropanol, and acetone maintain or slightly enhance enzyme activity, while methanol, N,N-dimethylformamide, and dithiothreitol inhibit enzyme activity. Among these, DTT shows the strongest inhibition, with a relative enzyme activity below 53%.

[0045] Example 4: Keratinase Sa Cut27 degrades shredded waste polyester. The enzyme was added to a 2‰ (m / m) substrate-to-enzyme buffer containing 5 g / L substrate in 4 mL PBS and 2 M sodium chloride. The reaction was carried out at 50 °C and pH 8.0 for 48 h. 100 µL samples were taken at 4, 8, 12, 24, 36 and 48 h, and 900 µL of methanol was added to terminate the reaction. The supernatant was collected by centrifugation and analyzed by HPLC. Figure 4 (A) Showing the control group under high salt conditions Is PETase and Sa Changes in the accumulation of PET degradation products by Cut27 over 0-48 h. Control group. Is PETase degradation was low, stabilizing after 12 h, with only 0.85 mM TPA and MHET produced at the reaction endpoint (48 h), corresponding to a degradation rate of approximately 3.2%; in contrast, Sa Cut27 maintained a higher product accumulation rate throughout the reaction, producing 3.07 mM of product at 24 h, which was the highest among similar reactions. Is 3-4 times that of PETase; at the 48-hour reaction endpoint, Sa The product concentration of Cut27 reached 4.17 mM, achieving a degradation rate of 17% ± 1.5%, which is 5–6 times that of the control group. This example can be used to infer the keratinase... Sa Cut27 exhibits halophilic and thermophilic properties, and shows better performance at high salinity and moderate temperatures than... Is PETase has better catalytic ability. Figure 4 (B) Display Is PETase and Sa Physical and electron micrographs of Cut27 and blank groups after 48 hours of reaction, showing that Sa Cut27 exhibits stronger corrosive and depolymerizing capabilities against this plastic. Overall, Sa Under these conditions, Cut27 is more effective than other depolymerization methods for crushed waste polyester. Is PETase.

[0046] Example 5: Keratinase Sa Construction of the Cut27 mutant library 1. PCR amplification according to Sa Based on the amino acid sequences of Cut27 and its mutants, upstream and downstream primers containing the mutation sites were designed (see Table 1 for details), and chemically synthesized by a primer synthesis company. Sa Using the plasmid of the Cut27 gene as a template, PCR amplification was performed using high-fidelity DNA polymerase. The reaction system was as follows: 25 μL of 2×Phanta Max Master Mix (Vazyme, catalog number: P515), 2 μL of upstream primer, 2 μL of downstream primer, 1 μL of template DNA, and double-distilled water to a final volume of 50 μL. PCR amplification conditions were: 95℃ pre-denaturation for 3 min; followed by 95℃ denaturation for 15 s, 65℃ annealing for 15 s, and 72℃ extension for 3 min, for 30 cycles; and a final incubation at 72℃ for 5 min.

[0047] 2. Dpn I restriction endonuclease digestion After PCR amplification, 1 mL of Dpn I restriction endonuclease was added directly to 50 mL of the amplification product. After thorough mixing, the mixture was incubated at 37°C for 5 min. Utilizing the specific recognition and cleavage of methylated template DNA by Dpn I, interference from wild-type plasmid templates was eliminated, thereby obtaining DNA containing the target mutation site. Sa Cut27 mutant plasmid.

[0048] 3. Transformation and Expression The digestion products were transformed into E. coli BL21(DE3) competent cells and plated on LB solid medium containing 50 μg / ml kanamycin (Kan). The cells were incubated upside down at 37°C for 12–16 h. Positive single colonies were picked and inoculated into LB liquid medium containing 50 μg / mL Kan, and cultured at 37°C with shaking at 220 rpm for 12 h. Seed culture was inoculated at a ratio of 1% (v / v) into TB liquid medium (containing 50 μg / mL Kan) and cultured at 37°C with shaking at 220 rpm. When the cell density (OD600) reached 0.6–0.8, IPTG was added to a final concentration of 0.1 mM, followed by induction at 18°C ​​for 20–24 h. After induction, the cells were collected by centrifugation (8000 rpm, 4°C, 10 min). The cells were then disrupted using an ultrasonic cell disruptor, and the supernatant was collected by centrifugation again. The supernatant was filtered through a membrane and then purified using a nickel-NTA (Ni-NTA) column. Gradient elutions were performed using elution buffers containing 50 mM, 100 mM, 200 mM, 300 mM, and 500 mM imidazole, respectively. The eluates were collected, and the target protein product and its purity were identified by SDS-PAGE gel electrophoresis. Finally, the target eluate was desalted and concentrated to obtain the purified protein. Sa Cut27 mutant protein.

[0049] 4. Sa Screening for Cut27 mutants The results obtained in Example 1 Sa The thermal stability, residual enzyme activity, and specific enzyme activity of Cut27 protein and the mutant protein obtained above were determined.

[0050] The thermal stability of the protein was evaluated using differential scanning calorimetry (DSC). The protein was dialyzed thoroughly in PBS buffer (pH 8.0), diluted to 1.0 mg / mL, filtered through a 0.22 μm filter, and degassed. Temperature scans were performed at a rate of 1.0 °C / min within the range of 25–95 °C, maintaining a system pressure of 2.5 atm. Using the same batch of dialysis buffer as a baseline control, a single symmetrical endothermic peak was selected, and the temperature corresponding to the peak of the endothermic peak was defined as the melting temperature (Tm). The results are shown in Table 2.

[0051] Residual enzyme activity assay: First measure Sa The initial enzyme activity of Cut27 and its mutants at pH 8.0 was measured, and then they were placed in pH 8.0 buffer and incubated at 50°C for 12 h. After the reaction, samples were taken to determine the residual enzyme activity. The results are shown in Table 2.

[0052] Enzyme activity calculation: Protein specific enzyme activity (U / mg) = Total enzyme activity of purified protein solution (U / mL) / Total protein content of purified protein solution (mg / mL). Sa Using Cut27 as a 100% reference, the percentage of specific enzyme activity of its mutants was calculated. The results are shown in Table 2.

[0053] As shown in Table 2, the specific enzyme activities of the three mutants S123E, S122Q, and H240Y compared to the wild type... Sa There is an improvement in Cut27; the specific enzyme activities of the three mutants S121G, L148F, and D260C are close to those of the wild type. Sa In the case of Cut27, the Tm value increased. Therefore, S123E, S122Q, H240Y, S121G, L148F, and D260C can be selected for subsequent screening of combined mutations.

[0054] Based on the above single-point mutations, a combined mutant was obtained. Using crushed waste polyester as a substrate (5 g / L), enzyme solution was added at an enzyme-to-substrate ratio of 2‰, along with 4 mL of a 2M pH 8.0 buffer solution. The reaction was carried out at 50°C. Figure 5 As shown, the combined mutants S121G / L148F / H240Y (T1), S121G / D260C / H240Y (T2), and L148F / D260C / H240Y (T3) exhibited significantly improved degradation performance compared to the wild type. Among them, the S121G / L148F / H240Y (T1) mutant showed the best degradation performance, with MHET+TPA production reaching 21.59 mM after 3 days of reaction, and a degradation rate of 88%.

[0055] Table 1 shows the primer information used. S121G -F CAcgcaggctcctcgCTCCTCGTCTATGGATTGGATGG S121G-R AGcgaggagcctgcgTGCGGTATAACCTGGCGC S121D-F CAccgcagactcctcgCTCCTCGTCTATGGATTGGATGG S121D-R AGcgaggagtctgcggTGCGGTATAACCTGGCGC [[ID=11{10]]S123E-F CTCCtccgaatctTCTATGGATTGGATGGGTCCG S123E-R TAGAagattcggaGGAGGATGCGGTATAACCTGG Q150G-F ATTAGGAAGaggggcccGCCCAGTAGTAGGTTGAGGCC Q150Y-R CgggcccctCTTCCTAATGCAGGAGTCGCA D260C-F TGGAACTGtgtgcggCGGCGCGAGCCATTTTGC D260C-R CGccgcacaCAGTTCCACGTACATACGATCGG S122Q-F ATCCtcccaatcgTCGTCTATGGATTGGATGGGTC S122Q-R ACGAcgattgggaGGATGCGGTATAACCTGGCG L148F-F ATACCCGTTTcGACCAGCCGAGCTCTCGCGGT L148F-R CTGGTCgAAACGGGTATTGGTATCAATGACAA I197A-F TTGGAGGCTACCgTCAGCCGCCCATCCTTGAA I197A-R CTGAcGGTAGCCTCCAACGCACCGCCGCCACC P210G-F CccggttggaTTGGAATCTGACCAAGAACTGGA P210G-R ATTCCAAtccaaccggGGTCAGCGGGATGGAGGC H240Y-F TGCGTACGtATAGCATCCCGTTCTATACCAGCC H240Y-R GATGCTATaCGTACGCACGGAGGCAACGGTAT Table 2 on Tm values, residual enzyme activity, and specific enzyme activity of Cut27 and mutants Cut27 52℃±0.12 51% 100% S121G 60℃±0.08 50% 96% S121D 58℃±0.04 24% 37% S123E 61℃±0.12 60% 102% Q150G 64℃±0.08 41% 84% D260C 72℃±0.06 48% 97% S122Q 54℃±0.06 59% 116% L148F 52℃±0.02 52% 105% I197A 61℃±0.17 37% 64% P210G 50℃±0.08 39% 71% H240Y 58℃±0.12 72% 145% Table 3 on Tm value, residual enzyme activity, and specific enzyme activity of the Cut27 combinatorial mutant S121G / L148F / H240Y (T1) 88℃±0.08 78% 154% S121G / D260C / H240Y (T2) 77℃±0.04 72% 143% L148F / D260C / H240Y (T3) 82℃±0.12 59% 136%

Claims

1. A keratinase mutant resistant to salt-hydrolyzed PET, characterized in that, The mutant is based on the wild-type keratinase SaCut27 with the amino acid sequence shown in SEQ ID NO.1, and is obtained by the following combination of mutations: S121G / D260C / H240Y.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the keratinase mutant of salt-tolerant hydrolyzed PET as described in claim 1.

3. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 3, or the nucleic acid molecule of claim 2 is integrated into its gene.

5. A method for preparing the keratinase mutant of salt-tolerant hydrolyzed PET as described in claim 1, characterized in that, The host cells described in claim 4 are cultured, and the expression product is separated and purified to obtain a salt-tolerant PET hydrolysate keratinase mutant.

6. The application of the keratinase mutant of salt-tolerant hydrolyzed PET as described in claim 1, the nucleic acid molecule as described in claim 2, the recombinant expression vector as described in claim 3, or the host cell as described in claim 4 in the depolymerization of PET plastic.

7. The application according to claim 6, characterized in that, The depolymerization of PET plastic is carried out in a salt-containing reaction system with a salt concentration of 1–5 M.

8. A method for depolymerizing PET plastic, characterized in that, In a salt-containing reaction system, the keratinase mutant of salt-tolerant hydrolyzable PET as described in claim 1 or the host cell as described in claim 4 is added to the plastic to cause the PET to undergo a hydrolysis reaction.

9. The method for depolymerizing PET plastic according to claim 8, characterized in that, The salt concentration in the reaction system is 2M, the reaction temperature is 50℃, and the reaction time is 12 h–72 h.

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