Hydrolase TmFae-PETase mutant capable of improving thermal stability and catalytic efficiency and application of hydrolase TmFae-PETase mutant in PET degradation

Abstract

The invention discloses a recombinant Tm Fae-PETase mutant and application of the recombinant Tm Fae-PETase mutant in PET hydrolysis, and belongs to the technical field of bioengineering. According to the transformation method, a series of high-performance mutants are designed through rational protein engineering, and the problem that the catalytic efficiency of wild Tm Fae-PETase is low is effectively solved. Wherein the specific enzyme activity and the thermal stability of the mutants I199D and V227I are obviously improved compared with those of wild enzymes; the specific enzyme activity of the mutant M198I (methionine at the 198 site is mutated into isoleucine) is improved to 365.289% of that of a wild type. The high-performance mutants significantly improve the degradation efficiency of PET waste, and provide a powerful core enzyme element for promoting the industrial process of biological recovery of PET.

Description

Technical Field

[0001] This invention relates to a hydrolytic enzyme that improves thermal stability and catalytic efficiency. Tm Fae-PETase mutants and their application in PET degradation belong to the field of bioengineering technology. Background Technology

[0002] The continuous accumulation of polyethylene terephthalate (PET) waste, which is difficult to degrade naturally, has become a serious environmental challenge. To address this issue, the academic community has successively developed various PET recycling technologies, including chemical, physical, and biological methods. Among them, enzymatic hydrolysis is considered a promising green recycling pathway due to its advantages such as environmental friendliness, low energy consumption, and low waste generation.

[0003] Currently, researchers have identified a total of 311 PET hydrolases from diverse ecosystems, including insect guts, human saliva, marine environments, and tropical forests. These mainly include esterases, lipases, carboxylesterases, and keratinases. Driven by computational methods, studies have been conducted on representative PET hydrolases (such as...). Is Significant progress has been made in protein engineering research on PETase and LCC, with the development of FAST-PETase, DuraPETase, DepoPETase, CaPETaseM9, and LCC. ICCG High-performance mutants are needed. However, existing PET hydrolases have relatively limited substrate catalytic activity. In previous work, the inventors screened and obtained a PET degrading enzyme with substrate heterogeneity, which has been disclosed in patent CN118374472A. However, the catalytic efficiency and / or thermal stability of this PET degrading enzyme still need improvement. Summary of the Invention

[0004] This invention provides a PET hydrolase with substrate heterogeneity. Tm Fae-PETase mutants and their application in PET degradation, derived from the intestines of yellow mealworms ( Tenebrio Molitor Larvae PET hydrolase Tm It was obtained by site-directed mutagenesis based on Fae-PETase (disclosed in patent CN118374472A).

[0005] In one embodiment, the mutant is obtained by mutating one or more of the amino acids at positions 25, 26, 27, 28, 129, 130, 133, 198, 199 and 227 of the amino acid sequence as shown in SEQ ID NO.1.

[0006] In one embodiment of the present invention, the mutant is any one of the following (a) to (k): (a) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The alanine at position 25 of Fae-PETase was mutated to aspartic acid, resulting in mutant A25D. (b) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The tyrosine residue at position 26 of Fae-PETase was mutated to phenylalanine, resulting in the mutant Y26F. (c) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The serine at position 27 of Fae-PETase was mutated to alanine to obtain mutant S27A. (d) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The glycine at position 28 of Fae-PETase was mutated to alanine, resulting in mutant G28A. (e) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The histidine at position 129 of Fae-PETase was mutated to asparagine, resulting in the mutant H129N. (f) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The valine at position 130 of Fae-PETase was mutated to alanine, resulting in mutant V130A. (g) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The asparagine at position 133 of Fae-PETase is mutated to serine, resulting in the mutant N133S. (h) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The methionine at position 198 of Fae-PETase was mutated to isoleucine, resulting in mutant M198I. (i) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The methionine at position 198 of Fae-PETase was mutated to leucine, resulting in mutant M198L. (j) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The isoleucine at position 199 of Fae-PETase was mutated to aspartic acid, resulting in mutant I199D. (k) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 TmThe valine at position 227 of Fae-PETase is mutated to isoleucine, resulting in mutant V227I.

[0007] The present invention also provides an enzyme encoding the above-mentioned PET hydrolase. Tm The gene of the Fae-PETase mutant.

[0008] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0009] In one embodiment, the recombinant vector uses pET-28a as the expression vector.

[0010] The present invention also provides microbial cells carrying the above-mentioned genes or the above-mentioned recombinant vectors.

[0011] In one embodiment, the microbial cells use prokaryotic cells as expression hosts.

[0012] In one embodiment, the microbial cells are... Escherichia coli Rosetta (DE3) is the expression host.

[0013] The present invention also provides a recombinant Escherichia coli that expresses the above-mentioned recombinant PET hydrolase. Tm Fae-PETase mutant.

[0014] In one embodiment, the recombinant Escherichia coli is used as Escherichia coli Rosetta (DE3) was used as the expression host, and pET-28a was used as the expression vector.

[0015] The present invention also provides a method for obtaining the above-mentioned recombinant PET hydrolase. Tm A method for Fae-PETase mutants, the method comprising the following steps: (1) In recombinant PET hydrolase Tm Based on the Fae-PETase amino acid sequence, the mutation site was determined; mutation primers were designed to carry recombinant PET hydrolase. Tm The Fae-PETase gene vector was used as a template for mutation; a plasmid vector with site mutation was constructed. (2) Transform the point-mutated plasmid vector into the host cell; (3) Select positive clones for culture and purify PET hydrolase. Tm Fae-PETase.

[0016] In one embodiment, the host cell is Escherichia coli.

[0017] The present invention also provides the application of the recombinant Escherichia coli or the method in the degradation of PET.

[0018] The present invention also provides a method for degrading PET, the method comprising: using the above-mentioned recombinant PET hydrolase Tm Fae-PETase mutant, or crude or pure enzyme expressed by the above-mentioned microbial cells or recombinant Escherichia coli, is added to a reaction system containing PET and reacted to prepare the product.

[0019] In one embodiment, the final concentration of PET in the reaction system is 1~5 mg / mL.

[0020] In one embodiment, the final concentration of the phosphate buffer in the reaction system is 50 mM.

[0021] In one embodiment, the PET hydrolase Tm The final concentration of the Fae-PETase mutant in the reaction system was 0.7–1.5 μM.

[0022] In one embodiment, the reaction conditions are: pH 6.5~8.5, 35~40°C, for 24~48 h.

[0023] The present invention also provides the above-mentioned recombinant PET hydrolase. Tm The application of Fae-PETase mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant Escherichia coli in PET hydrolysis or in the preparation of PET hydrolyzed products.

[0024] Beneficial effects: (1) This invention provides a recombinant PET hydrolase Tm Fae-PETase and its mutants are used to hydrolyze PET.

[0025] (2) The recombinant PET hydrolase of the present invention Tm The Fae-PETase mutant hydrolyzes PET into MHET and TPA, compared to the recombinant PET hydrolase. Tm Fae-PETase improves the production capacity and reaction efficiency of a unit catalyst, reduces reaction costs, and accelerates the industrialization of enzymatic hydrolysis of PET. Among them, mutant M198I, while maintaining stable thermal stability, exhibits a hydrolysis capacity 3.65 times that of the wild type; mutants I199D and V227I have melting points increased from 76.57℃ (wild type) to 86.50℃ and 84.38℃, respectively, with hydrolysis capacities increasing to 2.67 times and 2.05 times that of the wild type, respectively. Attached Figure Description

[0026] Figure 1 SDS-PAGE gel images of the mutant and wild-type enzymes.

[0027] Figure 2 Substrate heterogeneity of wild-type enzymes.

[0028] Figure 3 The ability of the mutant to hydrolyze PET in 24 hours.

[0029] Figure 4 The melting point temperature of the functionally enhanced mutant is increased. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0031] Reagents and materials: Prime STAR Max DNA polymerase, BamHI, HindIII, NdeI and XhoI endonucleases, DNA marker and other enzyme reagents were purchased from TaKaRa (Dalian).

[0032] The ClonExpress one-step directional cloning kit and gel extraction kit were purchased from Vazyme Biotech (Nanjing).

[0033] The plasmid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0034] PET, PCL, DEP, MSA, EA, PBS, PBT, and 4-HCMA refer to polyethylene terephthalate, polycaprolactone, diethyl phthalate, methanesulfonic acid, ethyl acetate, polybutylene succinate, polybutylene terephthalate, and 4-hydroxycinnamic acid, respectively. The PET was purchased from Goodfellow.

[0035] All analytical grade reagents were purchased from Sinopharm Group.

[0036] Culture medium: LB medium (g / L): yeast extract 5, NaCl 10, peptone 10; bring to the appropriate volume with deionized water, sterilize at 121 ℃ for 20 min before use; solid medium requires the addition of about 2% agar powder before sterilization.

[0037] TB medium (g / L): glycerol 4, yeast extract (Angel 802) 24, tryptone 12, KH2PO4 2.31, K2HPO4·3H2O 16.42; bring to the appropriate volume with deionized water, sterilize at 121 ℃ for 15-20 min and set aside for use.

[0038] PET hydrolase Tm In vitro catalytic reaction and product detection of Fae-PETase: Tm The degradation ability of Fae-PETase on PET plastic was assessed as follows: PET was prepared into powder, washed in ethanol, rinsed several times with ultrapure water, and then surface-sterilized with 75% ethanol. It was then dried at room temperature for later use. Preparation of the reaction solution: Phosphate buffer (K₂HPO₄ and KH₂PO₄, pH 8.0) prepared at 37 °C was used as the reaction solvent. A final concentration of 0.7–1.5 μM of [a specific solution] was added. Tm Fae-PETase was used, with the final concentration of PET in the reaction solution controlled at 5 mg / mL. All tests were repeated three times. The peak areas of PET degradation products TPA and MHET (1, 5, 10, 50, 100, 200 µM) at 240 nm were measured, and standard curves were constructed.

[0039] The hydrolytic capacity of PET hydrolase is measured by the total amount of TPA and MHET produced within a certain time.

[0040] The HPLC detection conditions are as follows: A C18 column (size: 4.6×250 mm) was used. The mobile phase A was 50 mmol / L phosphate buffer, the mobile phase B was methanol, the flow rate was 1.0 mL / min, the detector was a UV detector, the detection wavelength was 240 nm, the column temperature was 25 ℃, and the injection volume was 10 μL.

[0041] Example 1: Construction of wild-type genetically engineered bacteria Will Tm The Fae-PETase sequence was codon-optimized and synthesized artificially. The synthesized nucleotide sequence, as shown in SEQ ID NO.2, was then ligated and assembled into the pET28a (+) plasmid. Bam HI and Hin Between the d III restriction sites, pET28a (+)- is obtained. Tm Fae-PETase plasmid.

[0042] pET28a (+)- Tm Fae-PETase plasmid transformation E.coli Rosetta(DE3) competent cells were obtained to express... Fae-PETase's genetically engineered bacteria DE3- Tm Fae-PETase.

[0043] Example 2: PET hydrolase Tm Fae-PETase Substrate Confounding Test To expand TmBased on the substrate profile of Fae-PETase (this enzyme is known to hydrolyze PET, PBAT, methylferulate, methylcaffeate, and 4-nitrophenylbutyrate), we evaluated its activity against PCL, DEP, MSA, EA, PBS, PBT, and 4-HCMA. The specific experimental procedure included cell culture, enzyme purification, and subsequent enzyme activity assays, as follows: (1) Cell culture and preparation of crude enzyme solution Genetically engineered bacteria DE3- Tm A single Fae-PETase colony was inoculated into 30 mL of LB medium containing Kan and Cm resistance and cultured at 37°C with shaking for 10 h. The colony was then transferred to 150 mL of TB medium (containing the corresponding antibiotic) at a 1% (v / v) inoculation rate and cultured at 37°C and 200 rpm until OD500 was reached. 600 The concentration was set to 0.6–0.8, and IPTG was added to a final concentration of 60 mg / L. Expression was induced at 16°C for 18 h. After induction, the cells were collected by centrifugation at 4°C and 8000 rpm for 5 min. The cells were washed with PBS buffer and resuspended at a concentration of 10 mg / mL. The cells were then sonicated (100 W power, 2 s sonication, 1.5 s interval, total duration 10 min), and centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was then collected as the crude enzyme solution.

[0044] (2) Enzyme protein purification Purification was performed using a 5 mL Ni-NTA column. First, the column was equilibrated with binding buffer. After loading the crude enzyme solution, impurities were removed with wash buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole), followed by elution with elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole). The entire process was performed at 4°C. The eluted protein was concentrated and desalted using ultrafiltration (10 kDa molecular weight cutoff), and purity was assessed by SDS-PAGE. Protein concentration was determined using Nanodrop at 280 nm.

[0045] Mix 20 μL of protein (1 mg / mL) with 5 μL of 5×SDS-PAGE loading buffer (containing 2.5% β-methylethanol to a final concentration) and heat in a boiling water bath for 10 minutes to fully denature the protein. Perform electrophoresis using a 12% polyacrylamide separating gel and a 5% stacking gel. Load 20 μL per well, using a pre-stained protein molecular weight standard as a reference. Electrophoresis is performed in 1×Tris-Glycine SDS-PAGE buffer under the following conditions: constant voltage of 80 V during the stacking gel stage; after the sample enters the separating gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue front migrates to the bottom of the gel.

[0046] (3) Substrate activity assay Dissolve 0.5 g of PCL in 10 mL of chloroform (50-60 °C), add 0.01 g of Plysurf A210G, and bring the volume to 50 mL with ultrapure water. After ultrasonic emulsification, stir at 50 °C and 200 rpm for 2 hours to evaporate the chloroform. Bring the volume back to 50 mL to obtain a 10 mg / mL PCL substrate solution, and prepare a 1-10 mg / mL standard curve for quantitative analysis. Tm The degradation activity of Fae-PETase against PET, PBT, PBAT, and PBS was determined under the following conditions: reaction at 50°C for 24 hours, with the system containing 5 mg / mL plastic fragments, 50 mM phosphate buffer (pH 8.0), and 0.7 μM enzyme solution. DEP hydrolysis was performed in 50 mM Tris-HCl buffer (pH 8.0), containing 0.5 mM DEP and 0.7 μM enzyme solution, at 50°C and 180 rpm for 1 hour, followed by termination with 100 μL of 1M HCl. A240 calibration curves were plotted using 1–200 μM TPA, MHET, MEP, and SA standard solutions. Determination of MFA, MCA, MSA, EA, and 4-HCMA (1 mM) was performed in 50 mM phosphate buffer (pH 8.0), containing 0.7 μM enzyme solution, at 50°C for 24 hours. A320 calibration curves were plotted using 0.1–1.0 mM FA and CA standard solutions. The activity of p-NP(C4) was determined in 50 mM Tris-HCl buffer (pH 8.0) containing 2% acetonitrile, with 0.2 mM substrate and 0.7 μM enzyme solution. After reacting at 30 °C for 5 minutes, the absorbance at 405 nm was measured, and quantification was performed using a 0.1–1 mM p-nitrophenol standard curve.

[0047] The results of this invention Tm Fae-PETase exhibits catalytic activity against 12 classes of compounds, including PET, MFA, MCA, lignin, and lipids. Tm (As shown).

[0048] Example 3: Construction of mutant genetically engineered bacteria pET28a (+)- was prepared according to the method in Example 1. Figure 2 Fae-PETase plasmid.

[0049] design Tm Primers for the Fae-PETase mutant sites are shown in Table 1. Tm Using the Fae-PETase plasmid as a template, mutants were constructed by whole-plasmid PCR.

[0050] Table 1. Primer sequence listing for mutants

[0051] Constructing the PCR amplification system: Prime STAR Max DNA polymerase 25 μL, forward and reverse primers 1 μL each, template ( Tm 1 μL of Fae-PETase and 22 μL of water were used for the PCR reaction. The PCR reaction conditions were: ① 94 ℃ for 3 min; ② 98 ℃ for 10 s; ③ 58 ℃ for 5 s; ④ 72 ℃ for 10 s / kb; ⑤ Repeat steps ② to ④ 29 times; ⑥ 72 ℃ for 5 min; ⑦ Incubate at 12 ℃.

[0052] The above reaction system was incubated at 37 °C for 1 h to digest the plasmid template (the digestion system consisted of: 1 μL DpnI, 44 μL of the above reaction PCR product, and 5 μL 10×T Buffer). After digestion, the digestion product was introduced into Escherichia coli Rosetta competent cells by chemical transformation. The specific steps of the chemical transformation method were as follows: (1) 10 μL of homologous recombination product was introduced into 100 μL of... Tm Rosetta (DE3) competent cells; (2) Ice bath for 10 min; (3) Heat shock in a 42 ℃ water bath for 1 min 30 s, then quickly place in ice and let stand for 4 min; (4) Add 600 μL of antibiotic-free LB medium and mix well, then incubate at 37 ℃ and 200 rpm for 1 h; (5) Centrifuge at 5000 rpm for 2 min to collect the bacteria; (6) Remove the supernatant, and spread the remaining 100-200 μL onto LB resistant plates containing 0.05 mg / mL kanamycin, and incubate at 37 ℃ for about 12 h; (7) Pick single clones and incubate in LB containing 0.05 mg / mL kanamycin, incubate at 37 ℃ and 200 rpm for 12 h, then send to the company for sequencing. The ones with correct sequencing are positive transformants.

[0053] Following the method described in Example 2, engineered bacteria containing wild-type and mutant genes were cultured to induce target enzyme expression. After purification, purified samples of the wild-type enzyme and various mutant enzymes were obtained. SDS-PAGE analysis showed that the 11-point mutant exhibited a single, clear main band at approximately 29 kDa, and its migration position matched the theoretical molecular weight, indicating successful expression of the mutant protein in the *E. coli* system with good purity, suitable for subsequent functional studies. E.coli (As shown).

[0054] Example 4: Determination of the enhanced PET hydrolysis capacity of mutants Figure 1The degradation ability of Fae-PETase on PET plastic was tested as follows: PET was prepared into a 40-80 mesh powder, washed in ethanol, rinsed several times with ultrapure water, and then surface-sterilized with 75% ethanol. It was then dried at room temperature for later use. Preparation of the reaction solution: Phosphate buffer (K₂HPO₄ and KH₂PO₄, pH 8.0) prepared at 37 °C was used as the reaction solvent, and purified [agent] was added to a final concentration of 1 μM. Tm Fae-PETase or mutants were used, with the final concentration of Goodfellow-PET in the reaction solution controlled at 5 mg / mL. After 24 hours of reaction, the molar amounts of MHET and TPA generated by each mutant and wild type (WT) were quantitatively analyzed, and the product yield (sum of MHET and TPA) of each mutant was calculated as a multiple of the product yield obtained by the wild type, as the relative hydrolytic capacity. Tm As shown, this study successfully obtained several catalysts with significantly improved catalytic performance through site-directed mutagenesis technology. Figure 3 Fae-PETase mutants, particularly M198I, I199D, A25D, and V227I, exhibited catalytic efficiencies 3.7, 2.7, 1.2, and 2.1 times higher than the wild type, respectively, demonstrating significant application potential. These results lay a solid foundation for a deeper understanding of the structure-function relationship of PET hydrolases and their subsequent rational design.

[0055] Table 2. Catalytic activity of mutants on PET

[0056] Example 5: Determination of the improved thermal stability of mutants Accurately weigh the target protein, dilute it with pre-cooled 20 mM Tris-HCl buffer (pH 8.0), and bring it to a final working concentration of 0.5 mg / mL. A circular dichroism spectrometer equipped with a temperature control unit was used. The prepared sample was injected into a quartz cuvette with a 1 mm optical path. The method parameters were set as follows: detection wavelength 222 nm, temperature scan program linearly increasing from 30°C to 100°C at a rate of 2°C / min. The temperature scan program was started, and the instrument automatically recorded the CD value (usually expressed as ellipse intensity) at 222 nm as temperature increased. After the experiment, a graph was plotted with temperature on the x-axis and CD signal on the y-axis. The temperature corresponding to the inflection point of the curve was determined by fitting the curve; this was defined as the melting point (Tm) of the protein. The results are as follows: Tm As shown, this study successfully obtained several thermodynamically enhanced thermodynamic components through site-directed mutagenesis technology. Figure 4 Tm Fae-PETase mutants, especially I199D and V227I, have melting points that are increased from 76.57℃ in the wild type to 86.50℃ and 84.38℃, respectively.

[0057] Table 3 Melting point temperature of mutants

[0058] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. PET hydrolase Tm Fae-PETase mutant, characterized by It is obtained by mutating one or more of the amino acids at positions 25, 26, 27, 28, 129, 130, 133, 198, 199 and 227 of the amino acid sequence shown in SEQ ID NO.

1.

2. The hydrolytic enzyme according to claim 1 Tm Fae-PETase mutant, characterized by Having any of the mutations (a) to (d): (a) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The alanine at position 25 of Fae-PETase is mutated to aspartic acid; (b) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The methionine at position 198 of Fae-PETase is mutated to isoleucine. (c) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The isoleucine at position 199 of Fae-PETase is mutated to aspartic acid; (d) PET hydrolase with the amino acid sequence shown in SEQ ID NO.1 Tm The valine at position 227 of Fae-PETase is mutated to isoleucine.

3. Encoding the PET hydrolase as described in claim 1 or 2 Tm The gene of the Fae-PETase mutant.

4. A recombinant vector carrying the gene of claim 3.

5. Microbial cells carrying the gene of claim 3 or the recombinant vector of claim 4.

6. Recombinant Escherichia coli, characterized in that, The PET hydrolase described in claim 1 or 2 is expressed. Tm Fae-PETase mutant.

7. The recombinant Escherichia coli according to claim 6, characterized in that, by Escherichia coli Rosetta(DE3) was used as the expression host, and pET-28a was used as the expression vector.

8. A method for degrading PET, characterized in that, The PET hydrolase according to claim 1 or 2 Tm The Fae-PETase mutant, or the lysate of the microbial cell as described in claim 5, or the lysate of the recombinant Escherichia coli as described in claim 6 or 7, is added to a reaction system containing PET for reaction.

9. The method according to claim 8, characterized in that, The reaction should be carried out at 35-40°C for at least 24 hours.

10. The PET hydrolase according to claim 1 or 2 Tm The use of Fae-PETase mutant, or the gene of claim 3, or the recombinant vector of claim 4, or the microbial cell of claim 5, or the recombinant Escherichia coli of claim 6 or 7 in the degradation of PET or in the preparation of PET hydrolyzed products.

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