Cutinase mutant with improved polyester plastic degradation efficiency and application of cutinase mutant

Abstract

The invention discloses a cutinase mutant capable of improving polyester plastic degradation efficiency and application of the cutinase mutant, and belongs to the field of environmental science. According to the invention, a mutant ICCG from a branch and leaf compost metagenome source LCC is used as a parent enzyme, and one or more amino acids in the 48th site, the 75th site, the 100th site and the 247th site of the mutant ICCG are mutated to construct the mutant. Compared with a parent ICCG, the mutant M8-M14 constructed by the invention has the advantages that the efficiency of degrading polyester such as PET (Polyethylene Terephthalate), PTT (Polytrimethylene Terephthalate), PBT (Polybutylene Terephthalate) and PBAT (Polybutyleneadipate-Butylene Terephthalate) is remarkably improved, the highest degradation rates respectively reach 86.87%, 52.38%, 64.84% and 69.27%, and the mutant M8-M14 has a good industrial prospect.

Description

Technical Field

[0001] The invention relates to a cutinase mutant with improved degradation efficiency for polyester plastics and application thereof, belonging to the field of environmental science. Background Art

[0002] Polyester is a general term for polymers obtained by polycondensation of polyols and polyacids. The polyester materials that are widely used in production are mainly engineering plastics such as polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT) and polybutylene terephthalate-adipate (PBAT). However, the natural degradation rate of these polyester plastics in the environment after use and disposal is extremely low, and the long-term accumulation of their waste will cause serious environmental pollution. Therefore, countries around the world are actively exploring plastic degradation technology. Compared with traditional physical and chemical degradation methods (acid hydrolysis, alkaline hydrolysis, alcoholysis, thermal decomposition, etc.), biodegradation technology is a hot topic in current research. Cutinase is an ester bond hydrolase belonging to the α / β-hydrolase family, and its natural substrate is the natural polyester on the surface of plants. In recent years, many studies have proved that cutinase is also a highly potential synthetic polyester degrading enzyme. However, the degradation efficiency of existing cutinases on synthetic polyesters is still low, especially the degradation performance defects of crystalline polyesters with a high terephthalic acid content are large. Summary of the invention

[0003] The purpose of the present invention is to overcome the inefficiency of cutinase in degrading synthetic polyesters such as PET, PTT, PBT and PBAT, and to provide a new mutant of cutinase and its application. The present invention uses a mutant ICCG derived from the metagenome source LCC of foliage compost as a parent enzyme, and molecularly designs the parent enzyme by introducing positive charges in the active region and enhancing the binding of the side chains of amino acid residues, so as to increase the activity of the enzyme on the substrate PBAT.

[0004] The present invention provides a novel mutant of cutinase ICCG with improved degradation efficiency of polyester substrates, wherein the mutant comprises substitution of one or more amino acids at positions 48, 75, 100 and 247 corresponding to the polypeptide shown in SEQ ID NO:1.

[0005] In one embodiment, the substitution at position 48 is a substitution of arginine for serine.

[0006] In one embodiment, the substitution at position 75 is a substitution of valine with arginine.

[0007] In one embodiment, the substitution at position 100 is substitution of serine with arginine;

[0008] In one embodiment, the substitution at position 247 is a substitution of arginine for serine.

[0009] In one embodiment, the mutant is obtained by replacing serine with arginine at the 48th amino acid of the polypeptide shown in SEQ ID NO:1. The obtained mutant S48R is named M8, and its amino acid sequence is shown in SEQ ID NO:3.

[0010] In one embodiment, the mutant is obtained by replacing valine with arginine at the 75th amino acid of the polypeptide shown in SEQ ID NO:1. The obtained mutant V75R is named M9, and its amino acid sequence is shown in SEQ ID NO:4.

[0011] In one embodiment, the mutant is obtained by replacing serine with arginine at position 100 of the polypeptide shown in SEQ ID NO:1. The mutant S100R is named M10, and its amino acid sequence is shown in SEQ ID NO:5.

[0012] In one embodiment, the mutant is obtained by replacing serine with arginine at position 247 of the polypeptide shown in SEQ ID NO:1. The mutant S247R is named M11, and its amino acid sequence is shown in SEQ ID NO:6.

[0013] In one embodiment, the mutant is a mutant in which the 48th amino acid of the polypeptide shown in SEQ ID NO:1 is replaced by arginine for serine, and the 247th amino acid is replaced by arginine for serine. The obtained mutant S48R / S247R is named M12, and its amino acid sequence is shown in SEQ ID NO:7.

[0014] In one embodiment, the mutant is a polypeptide shown in SEQ ID NO:1 in which the 100th amino acid is replaced by arginine for serine, the 247th amino acid is replaced by arginine for serine, and the 218th amino acid is replaced by tyrosine for histidine. The obtained mutant S100R / S247R is named M13, and its amino acid sequence is shown in SEQ ID NO:8.

[0015] In one embodiment, the mutant is a mutant in which the 48th amino acid of the polypeptide shown in SEQ ID NO:1 is replaced by arginine for serine, the 75th amino acid is replaced by arginine for valine, and the 247th amino acid is replaced by arginine for serine. The obtained mutant S48R / V75R / S247R is named M14, and its amino acid sequence is shown in SEQ ID NO:9.

[0016] The present invention provides a polynucleotide encoding the mutant.

[0017] The present invention provides a nucleic acid construct or vector comprising the polynucleotide.

[0018] In one embodiment, the vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series vectors.

[0019] In one embodiment, the vector is pET24a, and the polynucleotide encoding the mutant is inserted into the multiple cloning restriction site of the pET24a vector.

[0020] The present invention provides a host cell expressing the mutant or comprising the polynucleotide.

[0021] In one embodiment, the host cell includes but is not limited to Escherichia coli, Pichia pastoris, Bacillus subtilis, and Saccharomyces cerevisiae.

[0022] The present invention provides a method for producing the Cutinase mutant.

[0023] In one embodiment, the method comprises:

[0024] a) culturing a host cell of the invention under conditions suitable for expressing the variant; and

[0025] b) optionally recovering the variant.

[0026] In one embodiment, the step a) is culturing the host cell in a culture medium containing a carbon source, a nitrogen source and an inorganic salt.

[0027] In one embodiment, a) is to culture the host cells in LB medium at 35-40°C and 150-250rpm until OD 600 =0.8±0.5, then add IPTG with a final concentration of 0.1 mM, and culture at 20-30°C and 150-250 rpm to obtain a fermentation broth.

[0028] In one embodiment, the step b) is to centrifuge the fermentation broth and take the supernatant to obtain a crude enzyme solution.

[0029] The present invention provides a product containing the Cutinase mutant.

[0030] In one embodiment, the product includes, but is not limited to, an enzyme preparation or composition comprising the Cutinase mutant.

[0031] In one embodiment, the enzyme preparation or composition contains an enzyme protectant; the protectant includes an acid-base regulator and a freeze-drying protectant.

[0032] The invention provides a method for degrading poly(butylene adipate / terephthalate) or a substance containing poly(butylene adipate / terephthalate). The method comprises adding the cutinase mutant into a system containing poly(butylene adipate / terephthalate) for reaction.

[0033] In one embodiment, the Cutinase mutant is added in an amount of not less than 0.6 mg / g substrate.

[0034] In one embodiment, the reaction is carried out at pH 8.0±0.5, 70-75° C., and 150-250 rpm.

[0035] In one embodiment, the reaction time is not less than 24 hours.

[0036] In one embodiment, the reaction time is not less than 48 hours.

[0037] The present invention provides the use of the mutant in degrading polyester plastics such as polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT) and polybutylene terephthalate-adipate (PBAT), as well as materials containing the above polyester components.

[0038] In one embodiment, the material containing polyesters such as polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT) and polybutylene terephthalate-adipate (PBAT) includes agricultural mulch films, straws, plastic bags and the like.

[0039] Beneficial effects: The present invention obtains a series of mutants by transforming mutant ICCG derived from the metagenome source LCC of twig and leaf compost, wherein the mutants undergo single-point or multi-point mutations near the substrate binding site of the ICCG, and mutate the 48th, 75th, 100th, and 247th positions thereof, thereby constructing 7 mutants. Compared with the parent ICCG, the 7 mutants of ICCG described in the present invention (M8-M14) have significantly improved the efficiency of degrading polyesters such as polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), and polybutylene terephthalate-adipate (PBAT), and have good industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the degradation effect of the parent ICCG and its mutants on PBAT.

[0041] Figure 2 This is the degradation effect of the parent ICCG and its mutants on PET.

[0042] Figure 3 The degradation effect of parent ICCG and its mutants on PTT.

[0043] Figure 4 The degradation effect of the parent ICCG and its mutants on PBT.

[0044] Figure 5 The fermentation process curves of mutant M14 (S48R / V75R / S247R) in 3L and 6-ton fermenters. DETAILED DESCRIPTION

[0045] The present invention will be further described below in conjunction with specific embodiments.

[0046] Materials and reagents involved:

[0047] Polyester plastics such as polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT) and polybutylene terephthalate-adipate (PBAT) involved in the following examples were purchased from Shanghai McLean Biochemical Technology Co., Ltd. Polyethylene terephthalate (PET) plastics were purchased from Goodfellow Company. Terephthalic acid (TPA) was purchased from Sigma Company.

[0048] The E. coli JM109 and E. coli BL21 (DE3) involved in the following examples were purchased from Takara Biotech (Beijing) Co., Ltd.

[0049] The culture media involved are as follows:

[0050] LB solid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, agar 13, pH 7.0.

[0051] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, pH 7.0.

[0052] Trace element solution (g·L -1 ): FeSO4·7H2O 10.0, ZnSO4·7H2O 2.2, CaCl2 2.0, CuSO4·5H2O 1.0, MnSO4·4H2O 0.5, Na2B4O7·10H2O 0.2, (NH4)6Mo7O 24 0.1.

[0053] Fermentation medium (g·L -1): (NH4)2HPO4 4.0, KH2PO4 13.5, MgSO4·7H2O 1.4, citric acid 1.7, industrial grade peptone 1.0, industrial grade yeast powder 2.0, glycerol 8.0, trace element solution 10.0mL.

[0054] Feed medium (g·L -1 ): Glycerol 500.0, MgSO4·7H2O 15.0, industrial grade peptone 2.0, industrial grade yeast powder 4.0.

[0055] Sequences and abbreviations involved:

[0056] M9 (V75R): Based on the parent, position 75 is substituted with arginine (R).

[0057] M10(S100R): Based on the parent, position 100 is substituted with arginine (R).

[0058] M11 (S247R): Based on the parent, position 247 is substituted with arginine (R).

[0059] M12 (S48R / S247R): Based on the parent, positions 48 and 247 were substituted with arginine (R) and arginine (R), respectively.

[0060] M13 (S100R / S247R): Based on the parent, positions 100 and 247 were substituted with arginine (R) and arginine (R), respectively.

[0061] M14 (S48R / V75R / S247R): Based on the parent, positions 48, 75, and 247 were substituted with arginine (R), arginine (R), and arginine (R), respectively.

[0062] In describing the variants of the invention, the nomenclature described below has been adapted for ease of reference to employ the accepted IUPAC single-letter or three-letter abbreviations for amino acids.

[0063] Substitutions: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid.

[0064] The cutinase of the present invention comprises a substitution corresponding to position 48 of the sequence shown in SEQ ID NO: 1, which is substituted with arginine (R); a substitution corresponding to position 75, which is substituted with arginine (R); a substitution corresponding to position 100, which is substituted with arginine (R); a substitution corresponding to position 247, which is substituted with arginine (R); wherein,

[0065] In one embodiment, the Cutinase, the substitution at position 48, is substituted with arginine (R);

[0066] In one embodiment, the Cutinase, the substitution at position 75 is substituted with arginine (R);

[0067] In one embodiment, the Cutinase, the substitution at position 100 is substituted with arginine (R);

[0068] In one embodiment, the Cutinase, the substitution at position 247, is substituted with arginine (R).

[0069] The detection methods involved are as follows:

[0070] Protein concentration determination method:

[0071] The protein concentration was determined by the Coomassie Brilliant Blue method (Analytical Biochemistry 1976 72(1-2):248-54).

[0072] Detection method of degradation products and their content:

[0073] Standard treatment: Weigh TPA, MHET, and BHET standards and dissolve them in dimethyl sulfoxide (DMSO) to prepare a mother solution. Dilute the mother solution with sterile water to a 0.1 mg / mL standard solution, filter with a 0.22 μM filter, and inject into a liquid phase bottle with a syringe for HPLC detection.

[0074] Sample treatment: After the reaction is completed, take 0.5 mL of the reaction solution, centrifuge and take the supernatant, add methanol to dilute, filter with a 0.22 μM filter head, inject into a liquid phase bottle with a syringe, and perform HPLC detection.

[0075] Enzyme activity assay method:

[0076] The activity of plastic-degrading enzymes was determined using the p-nitrophenylbutyrate (pNPB) method.

[0077] Drawing of standard curve: Prepare 100 μmol / L p-nitrophenol (pNP) stock solution with 10 mM pH 8.0 Tris-HCl buffer, and then dilute with Tris-HCl buffer to 0, 20, 40, 60, 80, 100 μmol / L. Measure the absorbance at a wavelength of 405 nm on a spectrophotometer using a 0.5 cm cuvette, and draw a standard curve A=a×C+b with pNP concentration C as the abscissa and absorbance value A as the ordinate.

[0078] Enzyme activity detection: Pipette 1.44mL 10mM pH 8.0 Tris-HCl buffer (pre-incubated at 60℃ for 30min) into a 0.5cm glass cuvette; Pipette 30μL of the diluted sample to be tested; Pipette 30μL 50mmol / L pNPB substrate solution, add it to the above quartz cuvette, shake well and immediately place it in a visible spectrophotometer; Measure the A value at the absorption wavelength of 405run, record the A value every 5 seconds, the reaction time is 1 minute, and then bring it into the standard curve to calculate the enzyme activity of the enzyme solution (unit U / mL). Enzyme activity definition: The amount of enzyme required to catalyze the hydrolysis of the substrate pNPB to generate 1μmol pNP per unit time is defined as one enzyme activity unit (U).

[0079] Enzyme activity (U / mL) = [[(Kb) × V1) / [a × V2 × 1000]] × N

[0080] Where: K: the slope of the curve formed by the A value and time (min) measured at different times of the enzyme reaction; V1: reaction volume (mL); V2: enzyme added (mL); N: dilution multiple.

[0081] HPLC determination method:

[0082] Chromatographic conditions: Agilent 1200HPLC chromatograph, Agilent autosampler, Athena C18-WP, 4.6mm×250mm, 5μm chromatographic column, Agilent UV detector, detection wavelength of 240nm, mobile phase of anhydrous methanol and 1% (mass volume ratio) acetic acid aqueous solution mixed at 35%:65%, column temperature set at 30℃, flow rate 0.5mL·min -1 .

[0083] Calculation method of PBAT degradation rate:

[0084] Degradation rate (%) = A / (B×C)×100;

[0085] A is the mass (mg) of TPA in the reaction system; B is the percentage of TPA determined when PBAT is fully degraded by 20% NaOH; C is the mass (mg) of PBAT in the reaction system.

[0086] Calculation method of PET degradation rate:

[0087] Degradation rate (%) = ((m1 / x1+m2 / x2+m3 / x3)×M) / (mass of PET before treatment)×100;

[0088] m1, m2 and m3 represent the weight of TPA, MHET and BHET in the volume of the reaction mixture; M is the relative molecular mass of the PET unit; x1 is the relative molecular mass of TPA; x2 is the relative molecular mass of MHET; x3 is the relative molecular mass of BHET.

[0089] Calculation method of PTT or PBT degradation rate:

[0090] Degradation rate (%) = ((m1 / x1) × M) / (PTT or PBT mass before treatment) × 100;

[0091] m1 represents the weight of TPA in the volume of the reaction mixture; M is the relative molecular mass of the PTT or PBT unit; x1 is the relative molecular mass of TPA.

[0092] Example 1: Construction and expression of ICCG mutants

[0093] (1) Construction of ICCG mutant recombinant plasmid

[0094] The parent cutinase (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 2) was inserted into the NdeⅠ site and XhoⅠ site of pET24a by restriction endonuclease to obtain pET24a-ICCG; then, using site-directed mutagenesis technology, using the pET24a-ICCG plasmid as a template, PCR was performed using the primers shown in Table 1 to obtain the target gene mutation fragment (primers are shown in Table 1). The target fragment was connected to the pET-24a expression vector by Megawhop PCR to obtain a recombinant plasmid. The constructed recombinant plasmid was transformed into Escherichia coli JM109 to obtain a transformation product; the transformation product was spread on LB solid culture medium (containing 30 μg / mL kanamycin), and inverted cultured in a 37°C constant temperature incubator for 8 to 12 hours to obtain transformants; the transformants were picked and inoculated into LB liquid culture medium, and after shaking flask culture at 37°C and 120 to 180 rpm for 8 to 12 hours, the plasmid was extracted for sequencing verification to obtain recombinant plasmids expressing different ICCG mutants.

[0095] Table 1 Primer sequences

[0096]

[0097] (2) Construction of ICCG mutant recombinant Escherichia coli

[0098] The correct recombinant plasmid obtained in step (1) is transformed into Escherichia coli BL21 to obtain a transformation product; the transformation product is spread on LB solid culture medium (containing 30 μg / mL kanamycin), and inverted in a constant temperature incubator at 37°C for 8-12 hours to obtain a transformant; the transformant is selected and inoculated into LB liquid culture medium, and shaken at 37°C and 120-180 rpm for 8-12 hours, after which the plasmid is extracted for enzyme digestion verification and sequencing verification. If the verification is correct, the recombinant Escherichia coli is obtained: E. coli BL21 / pET-24(+)-WT, E. coli BL21 / pET-24A(+)-M9, E. coli BL21 / pET-24(+)-M10, E. coli BL21 / pET-24(+)-M11, E. coli BL21 / pET-24(+)-M12, E. coli BL21 / pET-24(+)-M13, E.coli BL21 / pET-24(+)-M14; among them, mutant V75R is abbreviated as M9, mutant S100R is abbreviated as M10, mutant S247R is abbreviated as M11, mutant S48R / S247R is abbreviated as M12, mutant S100R / S247R is abbreviated as M13, and mutant S48R / V75R / S247R is abbreviated as M14.

[0099] (3) Expression of ICCG mutants

[0100] The ICCG mutant recombinant E. coli obtained in step (2) was transferred to 100 mL of LB liquid medium at a 5% (v / v) inoculation volume and cultured at 37°C, 200 rpm shaking for 3 h until the OD reached 0. 600 =0.8, then add IPTG to a final concentration of 0.1 mM, continue shaking culture at 25°C and 200 rpm for 24 hours to obtain fermentation broth; centrifuge at 8000 rpm for 15 minutes to obtain the supernatant, which is the crude enzyme solution. The enzyme activity of the crude enzyme solution was measured, and the results showed that the enzyme activities of WT, mutants M9 (V75R), M10 (S100R), M11 (S247R), M12 (S48R / S247R), M13 (S100R / S247R), and M14 (S48R / V75R / S247R) were 13.6, 11.8, 9.2, 16.2, 14.8, 11.9, and 10.7 U / mL, respectively.

[0101] Preparation of enzyme protein: After filtering the crude enzyme solution with a 0.22μm inorganic membrane, the protein was purified by nickel column affinity chromatography. The solutions required for purification include binding buffer and washing solution. The binding buffer contains 25mM Tris, 500mM sodium chloride, adjusted to pH 7.4; the washing solution contains 20mM Tris, 500mM sodium chloride, 300mM imidazole, adjusted to pH 7.4. Adjust the ratio of binding solution and washing solution, perform gradient elution, and use SDS-PAGE analysis to verify the correct elution concentration. Use 20% binding solution concentration to elute impurities and 100% binding solution concentration to elute the target protein. Finally, use 10kDa ultrafiltration tube for ultrafiltration, use 50mM sodium phosphate buffer for replacement, and concentrate to obtain purified enzyme solution.

[0102] Example 2: Degradation performance of ICCG mutants on PBAT

[0103] 100 mg of PBAT particles that had been crushed and passed through a 60-mesh sieve were added to the reaction flask, and the parent ICCG and the ICCG mutant obtained in Example 1 were added at an addition amount of 0.6 mg enzyme protein / g substrate; -1 The total reaction volume was 5 mL in a potassium phosphate buffer solution of pH 8.0, and the mixture was stirred in a constant temperature water bath shaker at 75 °C and 200 rpm for 24 h. Since PBAT continuously produces acid during the degradation process, 6 mol·L -1 The pH was kept stable by adding NaOH. After centrifugation, methanol was added to dilute the reaction solution, and finally the degradation rate was calculated by HPLC analysis.

[0104] The results are as follows Figure 1 As shown, the degradation efficiency of PBAT by the seven ICCG mutants was significantly higher than that of the parent ICCG protein, among which the ICCG mutant M14 (S48R / V75R / S247R) had a 24h degradation rate of PBAT of 69.27%, which was 10.14 times that of the parent.

[0105] Example 3: Degradation performance of ICCG mutants on PET

[0106] 100 mg of amorphous PET particles that had been crushed and passed through a 60-mesh sieve were added to the reaction flask, and the parent ICCG and the ICCG mutant obtained in Example 1 were added at an addition amount of 0.6 mg enzyme protein / g substrate; -1 The total reaction volume was 5 mL in a potassium phosphate buffer solution of pH 8.0, and the mixture was stirred in a constant temperature water bath shaker at 65°C and 200 rpm for 12 h. Since PET continuously produces acid during the degradation process, 6 mol·L -1The pH was kept stable by adding NaOH. After centrifugation, methanol was added to dilute the reaction solution, and finally the degradation rate was calculated by HPLC analysis.

[0107] The results are as follows Figure 2 As shown, the degradation efficiency of PET by the seven ICCG mutants was significantly higher than that of the parent ICCG protein, among which the ICCG mutant M14 (S48R / V75R / S247R) had a PET degradation rate of 86.87% in 12 h, which was 2.05 times that of the parent.

[0108] Example 4: Degradation performance of ICCG mutants on PTT

[0109] 100 mg of amorphous PTT particles that had been crushed and passed through a 60-mesh sieve were added to the reaction flask, and the parent ICCG and the ICCG mutant obtained in Example 1 were added at an addition amount of 0.6 mg enzyme protein / g substrate; -1 The total reaction volume was 5 mL in a potassium phosphate buffer solution of pH 8.0 and the mixture was stirred in a constant temperature water bath shaker at 65°C and 200 rpm for 48 h. Since PTT continuously produces acid during the degradation process, 6 mol·L -1 The pH was kept stable by adding NaOH. After centrifugation, methanol was added to dilute the reaction solution, and finally the degradation rate was calculated by HPLC analysis.

[0110] The results are as follows Figure 3 As shown, the degradation efficiency of PTT by the seven ICCG mutants was significantly higher than that of the parent ICCG protein, among which the ICCG mutant M14 (S48R / V75R / S247R) had a PET degradation rate of 52.38% in 12 hours, which was 9.94 times that of the parent.

[0111] Example 5: Degradation performance of ICCG mutants on PBT

[0112] 100 mg of amorphous PBT particles that had been crushed and passed through a 60-mesh sieve were added to the reaction flask, and the parent ICCG and the ICCG mutant obtained in Example 1 were added at an addition amount of 0.6 mg enzyme protein / g substrate; -1 The total reaction volume was 5 mL in a potassium phosphate buffer solution of pH 8.0 and the mixture was stirred in a constant temperature water bath shaker at 65°C and 200 rpm for 48 h. Since PBT continuously produces acid during the degradation process, 6 mol·L -1 The pH was kept stable by adding NaOH. After centrifugation, methanol was added to dilute the reaction solution, and finally the degradation rate was calculated by HPLC analysis.

[0113] The results are as follows Figure 4As shown, the degradation efficiency of PBT by the seven ICCG mutants was significantly higher than that of the parent ICCG protein, among which the ICCG mutant M14 (S48R / V75R / S247R) had a PET degradation rate of 64.84% in 12 h, which was 8.44 times that of the parent.

[0114] Example 6: Scale-up fermentation preparation of ICCG mutant M14 (S48R / V75R / S247R)

[0115] The recombinant E. coli BL21 / pET-24(+)-M14 constructed in Example 1 was transferred to 50 mL of industrial-grade LB medium at a 2% inoculation volume fraction, and a final concentration of 0.1 g·L -1 of ampicillin, 37°C, 200 r·min -1 Culture for 8 hours. Take the above seeds at an inoculation volume fraction of 10%, transfer them to a 3L fermenter, and the initial liquid volume is 1L (including 900mL of fermentation medium and 100mL of seed liquid). The growth temperature is maintained at 37°C through the instrument's own control, the dissolved oxygen is maintained at around 30% by adjusting the rotation speed and changing the oxygen introduction ratio, and the pH is maintained at 7.0 by adding ammonia water. After 5-7 hours of culture, the carbon source in the culture medium in the upper tank is exhausted, and the dissolved oxygen rebounds. A final concentration of 0.1mMIPTG is added to start induced fermentation. Thereafter, feed culture medium is added to the fermenter in an exponential manner until the end of fermentation. The specific growth rate is controlled at μ=0.2h by adjusting the feed rate and dissolved oxygen dO2. -1 . After 6-8 hours of feeding, the induction stage begins when the bacteria grow to a specific stage. The induction time, induction temperature and inducer dosage are determined by the corresponding fermentation strategy. After induction, samples are taken at regular intervals to measure the biomass and enzyme activity of the samples. The fermentation is terminated when the enzyme activity shows a significant downward trend.

[0116] The 6-ton tank fermentation adopts a similar process to the 3L tank. Compared with the 3L tank, the 6-ton tank uses manual control to increase or decrease the speed to ensure that the dissolved oxygen dO2 is maintained at 30% (20-40%), and the exponential feed changes the flow rate every 0.5h. Due to the speed limit of the large tank, the glycerol feed is appropriately limited to ensure the dissolved oxygen level during the fermentation process.

[0117] The fermentation results are as follows Figure 5 As shown, the fermentation period of mutant M14 (S48R / V75R / S247R) in 3L and 6-ton tanks was 27h. Among them, the highest DCW in 3L tank fermentation was 38.8g / L, the highest cutinase activity was 685.2U / mL, and the target protein content was 5.71g / L; the highest DCW in 6-ton tank fermentation was 39.3g / L, the highest cutinase activity was 663.1U / mL, and the target protein content was 5.53g / L.

[0118] Example 7: Composition or enzyme preparation containing ICCG mutant

[0119] The cutinase enzyme protein was prepared according to the method of Example 1, and the enzyme protein was mixed with an enzyme protective agent; the protective agent included an acid-base regulator and a freeze-drying protective agent.

[0120] The acid-base regulator is a conventional buffer that can maintain pH, such as malic acid-sodium malate buffer, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, Tris-hydrochloric acid buffer. The buffer is used to maintain the cutinase protein at its optimal pH condition to ensure its maximum catalytic activity.

[0121] The lyoprotectants are used to reduce the chemical and / or physical instability of the cutinase enzyme protein during freeze-drying and / or subsequent storage, including but not limited to sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannitol; amino acids, such as arginine or histidine; soluble salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly (ethylene glycol) or poly (propylene glycol); and combinations of any of the above.

[0122] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A cutinase mutant, characterized in that Based on the parent shown in SEQ ID NO: 1, one or more amino acids at positions 48, 75, 100 and 247 are substituted.

2. The cutinase mutant according to claim 1, characterized in that Having one or more mutations from (a) to (d): (a) mutating serine at position 48 to arginine; (b) mutating valine at position 75 to arginine; (c) mutating the serine at position 100 to arginine; (d) The serine at position 247 was mutated to arginine.

3. A polynucleotide encoding the mutant according to claim 1 or 2.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell expressing the mutant according to claim 1 or 2, or comprising the polynucleotide according to claim 3.

6. A method for preparing the cutinase mutant according to claim 1 or 2, characterized in that: The method comprises: a) culturing a host cell of the invention under conditions suitable for expressing the variant; and b) optionally recovering the variant.

7. A product containing the Cutinase mutant according to claim 1 or 2.

8. A method for degrading polybutylene adipate / terephthalate or a substance containing polybutylene adipate / terephthalate, characterized in that: The cutinase mutant according to claim 1 or 2 is added into a system containing poly(butylene adipate) / terephthalate to carry out a reaction.

9. The method according to claim 8, characterized in that The addition amount of the cutinase mutant is not less than 0.6 mg / g substrate; and the reaction is carried out at 70-75° C. and 150-250 rpm.

10. Use of the mutant according to claim 1 or 2 in degrading polyester plastics, characterized in that: The polyester plastic includes, but is not limited to, polyethylene terephthalate, polytrimethylene terephthalate, polybutylene terephthalate, and polybutylene terephthalate-adipate.

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