PET hydrolase mutant with improved thermal stability and application thereof

Abstract

The invention discloses a PET hydrolase mutant with improved thermal stability and application thereof, and belongs to the field of enzyme engineering. Starting from HotPETase, research is carried out based on deep learning design, enzyme engineering optimization and application. 10 to-be-verified single-point mutants capable of improving the enzyme performance are selected through model prediction in combination with manual evaluation, and then experimental screening verification is carried out. The method comprises the following steps: comprehensively selecting better single-point mutants and combined mutants, and evaluating by taking an amorphous PET (Polyethylene Terephthalate) film (with the crystallinity of 6.7%) as a substrate to obtain dominant mutants. Candidate mutants are efficiently expressed and purified based on cSAT 2.0, the enzyme activity of the optimal mutant Y63F is equivalent to that of HotPETase, and the thermal stability of the optimal mutant Y63F is 1.5 times that of HotPETase after the optimal mutant Y63F is subjected to heat treatment at 65 DEG C for 2 hours. The optimal mutant Y63F is subjected to a protein melting temperature (Tm) test, and the result shows that the Tm is increased by 2.5 DEG C.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering, and particularly relates to a PET hydrolytic enzyme mutant with improved thermal stability and application thereof. BACKGROUND

[0002] More than 400 million tons of plastic products are consumed annually, and it is extremely difficult for them to be depolymerized in the natural environment due to their stable structure, leading to increasingly serious global plastic pollution. Among them, polyethylene terephthalate (PET) is the most widely consumed polyester plastic. In recent years, enzymatic hydrolysis of plastics has become a very promising strategy in the field of plastic degradation due to its mild reaction conditions, low energy consumption, and easy product recovery. Previous studies have found that a hydrolytic enzyme IsPETase (Yoshida S., Hiraga K., Takehana T., et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science. 2016, 351: 1196-1199.) derived from Ideonella sakaiensis has high specificity for PET and can almost completely degrade PET to generate Bis(2-hydroxyethyl) terephthalate (BHET), Mono(2-hydroxyethyl) terephthalate (MHET), Terephthalic acid (TPA), and Ethylene glycol (EG). However, its low catalytic rate and poor thermal stability limit its application in industrial fields. Therefore, researchers have used enzyme engineering strategies such as directed evolution, rational design, and machine learning to obtain mutants with improved catalytic activity and thermal stability. Among them, HotPETase (Bell E.L., Smithson R., Kilbride S., et al. Directed evolution of an efficient and thermostable PET depolymerase. Nat Catalysis. 2022, 5: 673.) is a representative mutant that has improved activity and stability compared to the natural enzyme and has become an important tool in the current enzymatic degradation of PET technology. However, HotPETase still has poor stability in certain high-temperature environments, and its performance needs to be further optimized.

[0003] Therefore, the present application is proposed. SUMMARY

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a PET hydrolase mutant with improved thermal stability and its application. The present application combines deep learning model design and enzyme engineering optimization strategy to develop a high-performance PET hydrolase mutant that is more suitable for industrial application requirements.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] A PET hydrolase mutant with improved thermal stability, whose amino acid sequence is SEQ ID NO. 1, is obtained by any one of the following mutations:

[0007] Y63F, S124A, S136A, S166A, S169A, N172Q, C241N, T279S, Y63F / S124A, Y63F / C241N, S124A / C241N or Y63F / S124A / C241N; wherein Y63F, i.e. the 63rd amino acid is mutated from Y to F, and the others are the same;

[0008] A coding gene of the mutant.

[0009] The biological material related to the above-mentioned mutant is any one or a combination of the following biological materials:

[0010] (1) an expression cassette containing the above-mentioned coding gene;

[0011] (2) a recombinant expression vector containing the above-mentioned coding gene;

[0012] (3) a recombinant expression vector containing the expression cassette in (1);

[0013] (4) a recombinant bacteria containing the above-mentioned coding gene;

[0014] (5) a recombinant bacteria containing the expression cassette in (1);

[0015] (6) a recombinant bacteria containing the recombinant expression vector in (2) or (3).

[0016] Further, the starting vector of the recombinant expression vector in (2) and (3) is a pET series vector or a pPICZ alpha vector, etc.; preferably a pET-22b(+) vector or a pPICZ alpha A vector.

[0017] Further, the host bacteria corresponding to the recombinant bacteria in (4), (5), and (6) are selected from prokaryotes, yeasts, or higher eukaryotic cells, etc.; the prokaryotes include bacteria such as Escherichia, Bacillus, Salmonella, Pseudomonas, or Streptomyces; and the yeasts include yeasts such as Pichia. More specifically, the prokaryotes are Escherichia, preferably Escherichia coli (E. coli), and specifically can be E. coli BL21 (DE3) or E. coli Origami 2 (DE3); and the yeasts are Pichia X33 or GS115.

[0018] The mutant, the coding gene, and the biological material related to the mutant are used for preparing the PET hydrolase mutant with improved thermal stability.

[0019] Further, the mutant, the coding gene, and the biological material related to the mutant are used for one of the following applications:

[0020] (a) application in degrading PET;

[0021] (b) application in plastic degradation.

[0022] Preferably, the crystallinity of the PET is 20% or less, further 5-20%, and still further 6-8%; and still further 6.7%.

[0023] A method for obtaining the mutant includes the following steps: a gene encoding a PET hydrolase with an amino acid sequence as shown in SEQ ID NO. 1 is subjected to site-directed mutation by a site-directed mutation technique, and then expressed to obtain the PET hydrolase mutant with improved thermal stability.

[0024] Further, a mutation is introduced into a gene encoding a PET hydrolase with an amino acid sequence as shown in SEQ ID NO. 1 by a site-directed mutation technique, and then transformed into E. coli for expression after sequencing to obtain the PET hydrolase mutant with improved thermal stability.

[0025] The present application has the following advantages and effects relative to the prior art:

[0026] The present application is based on the design of deep learning, enzyme engineering optimization and application research starting from HotPETase. First, 10 single-point mutants that may improve enzyme performance are selected by using ISS-PMP deep learning model prediction combined with artificial evaluation, and then experimental verification and enzyme engineering modification are carried out. The better single-point mutants and combined mutants are selected and evaluated with amorphous PET film (amoPET, crystallinity 6.7%) as substrate to obtain the superior mutant. Based on the high-efficiency expression and purification of the superior mutant by the cleavable self-aggregation tag method (cSAT 2.0), the optimal mutant Y63F has an enzyme activity comparable to that of HotPETase, and the thermal stability after 2 hours of heat treatment at 65℃ is 1.5 times that of HotPETase. The protein melting temperature (T m ) test of the optimal mutant Y63F shows that its T m is increased by about 2.5℃. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the thermal treatment before and after the enzyme activity of the superior mutant and its melting temperature under the enzyme load of 0.29 mg / g PET.

[0028] Figure 2 is the result graph (A) of the TPA+MHET+BHET yield of the single-point mutant before thermal treatment and its residual activity (B) after thermal treatment under the enzyme load of 0.29 mg / g PET.

[0029] Figure 3 is the result graph (A) of the TPA+MHET+BHET yield of the combined mutant before thermal treatment and its residual activity (B) after thermal treatment under the enzyme load of 0.29 mg / g PET. DETAILED DESCRIPTION

[0030] The present application will be further described in detail below in conjunction with the examples and drawings, but the embodiments of the present application are not limited thereto. The test methods in the following examples are generally carried out according to the conventional experimental conditions or according to the experimental conditions suggested by the manufacturer, unless otherwise specified. The materials, reagents, etc. used are commercially available reagents and materials, unless otherwise specified.

[0031] Example 1: Construction of PET hydrolytic enzyme (PETase) single-point mutant

[0032] The PET hydrolytic enzyme (PETase) used in the example is HotPETase, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of the codon-optimized encoding gene is shown as SEQ ID NO. 2, which is synthesized by a commercial company.

[0033] 1. Obtain single point mutants based on ISS-PMP deep learning model

[0034] First, the predicted mutation sites and amino acids of the ISS-PMP deep learning model (software name: Protein Unit Point Mutation Design Software, registration number: 2024SR0792862) are sorted in descending order of probability.

[0035] Secondly, based on the above prediction results and combined with artificial evaluation, the following mutants (a total of 10) are selected as the single point mutants to be verified R207T, S169A, Y63F, T279S, C241N, S166A, N172Q, S124A, A152S, S136A.

[0036] 2. Construction and verification of single point mutant recombinant plasmid

[0037] (1) Recombinant plasmid cloning

[0038] The recombinant plasmid pET-22b(+)-HotPETase nucleotide sequence is used as a template, and full plasmid PCR polymerase chain reaction is performed with primers containing mutation sites to construct single mutant recombinant plasmids. Among them, the recombinant plasmid pET-22b(+)-HotPETase is constructed by inserting the nucleotide sequence (SEQ ID NO. 2, 82-870 bp) of the gene encoding HotPETase (SEQ ID NO. 1, 28-290 aa) into the NdeI to XhoI site between the multiple cloning site of the pET-22b(+) vector.

[0039] The PCR reaction system is 2xKOD buffer 25μL, 2.5mM dNTP 10μL, 1.0μL of each upstream and downstream primer, 10ng of plasmid template, 1μL of KOD enzyme, and ddH2O added to a final system of 50μL. The reaction conditions are 98℃ for 3min; 98℃ for 10s, 61℃ for 30s, 68℃ for 3min 30s, 30 cycles; 68℃ for 2min; 4℃ storage.

[0040] (2) Product assembly

[0041] The above product is assembled according to 25fmol of each DNA fragment, Gibson assembly Premix 15μL, ddH2O to 20μL. 50℃ reaction for 1h.

[0042] (3) Recombinant plasmid transformation and verification

[0043] Recombinant plasmid transformation: 10 μΐ of the above assembled ligation product was transformed into 100 μΐ of E. coli DH5a competent cells by chemical transformation, spread on LB solid plates containing a final concentration of 100 μg / mL carbenicillin, and incubated at 37°C overnight for about 12-16 hours.

[0044] Positive monoclonal verification: randomly pick 3 colonies of the monoclonal with positive PCR verification results for sequencing verification.

[0045] 3. Expression and purification of single-point mutant based on cSAT 2.0 method

[0046] Recombinant construction of PET hydrolytic enzyme single-point mutant based on cSAT 2.0 method to obtain a single-point mutant recombinant expression plasmid containing a cSAT 2.0 (Cleavable self-aggregating tag 2.0) skeleton. In the construction process, the signal peptide of the target gene HotPETase and the mutant needs to be removed, the amino acid sequence of which is shown as SEQ ID NO. 1 2-27 aa, and the nucleotide sequence encoding the signal peptide is shown as SEQ ID NO. 2 4-81 bp.

[0047] Take 5 μΐ of single-point mutant recombinant expression plasmid based on cSAT 2.0 method and transform into 100 μΐ of E. coli Origami2(DE3) competent cells, spread on LB solid plates containing a final concentration of 100 μg / mL carbenicillin, and incubate in a 37°C constant temperature incubator for 12-16 hours. Pick the colonies, and after positive colony PCR verification, streak the strain containing the target plasmid on LB plates with corresponding antibiotic resistance, and incubate at 37°C overnight.

[0048] Transfer the well-grown single colony to 5 mL of liquid LB medium containing 100 μg / mL carbenicillin, and incubate at 37°C, 220 rpm for 18 hours. Transfer 1 mL of seed liquid to 50 mL of fresh liquid 2xYT medium containing 100 μg / mL carbenicillin, and incubate at 35°C, 200 rpm until the OD 600 reaches 0.6-0.8. Induce protein expression with a final concentration of 0.2 mM IPTG, and further incubate at 18°C and 200 rpm for 20 hours. Centrifuge at 4,000 rpm for 20 min to collect the cSAT 2.0 bacterial precipitate.

[0049] 2xYT liquid medium preparation: 1% Yeast Extract, 1.6% Tryptone, 0.5% NaCl, dissolved in distilled water; 121°C, 20 min, high-pressure sterilization.

[0050] (3) Purification of single-point mutants based on the cSAT 2.0 method. SDS-PAGE electrophoresis was used for detection, thereby obtaining the purified candidate single-point mutants.

[0051] wherein the cSAT 2.0 method is disclosed in the literature “Huang Y, Zhang Y, Yang X, et al. A high-performance protein preparation approach in a single column-free step [J]. Trends in biotechnology: S0167-7799(24)00290-7. DOI: 10.1016 / j.tibtech.2024.10.008.”

[0052] Example 2: Screening of PET hydrolase single-point mutants

[0053] For the 10 candidate single-point mutants purified by the cSAT 2.0 method, the enzyme activity of the free enzyme was measured after reacting with the PET film substrate at 65°C for 3h (denoted as enzyme activity before heat treatment) and the residual enzyme activity after heat treatment for 2h and then reacting for another 3h (denoted as enzyme activity after heat treatment). Before heat treatment, the mutant enzyme activity was compared with the HotPETase enzyme activity to obtain the relative enzyme activity of each mutant. By comparing the enzyme activity after heat treatment with the enzyme activity before heat treatment, the residual activity was calculated; the residual activity of the mutant was compared with the residual activity of HotPETase to obtain the relative thermal stability, see Table 1 and Figure 2 . From Table 1 and Figure 2 it can be seen that compared with HotPETase, the thermal stability of single-point mutants Y63F, S124A and C241N is further improved, and the enzyme activity is retained by more than 80% or even equivalent.

[0054] PETase enzyme activity assay method for degrading substrate PET film: Amorphous PET film (amoPET, crystallinity 6.7%, Goodfellow, ES301445) was used as substrate, which was first made into a disc with a diameter of 6 mm and a rough weight of 8.5 mg, then soaked in 1% SDS solution, 20% ethanol solution and deionized water respectively for 30 min, and naturally air-dried as the actual substrate for PETase hydrolysis activity test. In a 96-deep-well plate, the total volume of glycine buffer (50 mM, pH 9.2) was 300 μL, the enzyme loading was 0.29 mg / g PET, the pretreated amoPET film was added, and the hydrolysis reaction was carried out at 65°C for 3 h. After the reaction was completed, an equal volume (300 μL) of cold methanol solution containing 12.5 mM trifluoroacetic acid was added to each reaction system, and the reaction was terminated at 30°C for 30 min. Then the yield of PET degradation products terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET) and bis(2-hydroxyethyl) terephthalate (BHET) was analyzed by high performance liquid chromatography (HPLC). HPLC analysis was completed by Agilent 1260 high performance liquid chromatograph coupled with Finnigan XB-C18 chromatographic column (2.6 μm, 50 x 2.1 mm, LC Column). The mobile phase A was water containing 0.1% formic acid, and the mobile phase B was 100% acetonitrile. The flow rate was set at 0.5 mL / min. The initial elution condition was 13% B to separate TPA and MHET, then the gradient was increased to 95% B, and finally the column was re-equilibrated to 13% B. The yield of the three products was calculated by comparing the product peak area with the corresponding standard. XB-C18 chromatographic column 2.6 μm, 50 x 2.1 mm, LC Column) was used to complete the identification. The mobile phase A was water containing 0.1% formic acid, and the mobile phase B was 100% acetonitrile. The flow rate was set at 0.5 mL / min. The initial elution condition was 13% B to separate TPA and MHET, then the gradient was increased to 95% B, and finally the column was re-equilibrated to 13% B. The yield of the three products was calculated by comparing the product peak area with the corresponding standard.

[0055] Thermal stability determination method of PETase degrading PET film: Pretreated 6 mm amorphous PET film (crystallinity 6.7%) was used as substrate. In a 96-deep-well plate, the total volume of glycine buffer (50 mM, pH 9.2) was 300 μL, the enzyme loading was 0.29 mg / g PET, and after 2 h of heat treatment at 65°C, amoPET film was added and the reaction was continued for 3 h. After the reaction was completed, an equal volume (300 μL) of cold methanol and trifluoroacetic acid mixture was added to each reaction system as a reaction termination solution to precipitate unreacted macromolecules and enzyme proteins, thereby removing potential analytical interferents. HPLC analysis was performed according to the method described above.

[0056] Table 1 Relative enzyme activity and relative thermal stability of single point mutants after 2 h of heat treatment at 65°C

[0057] Position Original amino acid Mutant amino acid Relative enzyme activity Residual activity Relative thermostability Hot PETase — — 1.00 0.58 1.00 Y63F Y(tat) F(ttt) 0.93 0.89 1.53 S124A S(agc) A(gcc) 0.89 0.76 1.30 S136A S(agc) A(gcc) 0.71 0.68 1.16 A152S A(gcc) S(agt) 0.36 0.01 — S166A S(tct) A(gcc) 0.58 0.71 1.22 S169A S(tcg) A(gcc) 0.68 0.87 1.49 N172Q N(aat) Q(cag) 0.70 0.73 1.25 R207T R(agg) T(acg) 0.07 0.39 — C241N C(tgt) N(aat) 0.80 0.75 1.26 T279S T(acc) S(agt) 0.59 0.71 1.22

[0058] For the above 10 single-point mutants, the enzyme activity after heat treatment at 65℃ for 2 hours and the enzyme activity before heat treatment were measured and compared with HotPETase. Eight single-point mutants with improved thermostability were screened out. Then, single-point mutants with improved thermostability and relative enzyme activity greater than 0.75 were screened out, and finally three single-point mutants with improved thermostability and relative enzyme activity greater than 0.75 were obtained (Table 1).

[0059] Example 3: Construction of combinatorial mutations of PET hydrolase

[0060] The three beneficial single-point mutants from Example 2 were randomly combined. Using the constructed plasmid containing the cSAT 2.0 backbone and the single-point mutant as templates, four corresponding combined mutant recombinant expression plasmids containing the cSAT 2.0 backbone were constructed using the Gibson assembly method.

[0061] The mutant construction and induced expression techniques described in Example 1 were followed, and the mutant enzyme activity and thermostability determination methods described in Example 2 were employed. The results are as follows: Figure 3 As shown in Table 2, the relative thermostability of the combined mutants was improved; subsequently, three beneficial combined mutants were screened out: Y63F / S124A, Y63F / C241N, and S124A / C241N, with relative enzyme activities greater than 0.75 and relative thermostabilities of 1.24, 1.22, and 1.20, respectively.

[0062] Table 2. Relative enzyme activity and relative thermal stability of the combined mutants after heat treatment at 65℃ for 2 h.

[0063] Position Relative enzyme activity Residual activity Relative thermostability Hot PETase 1.00 0.65 1.00 Y63F / S124A 0.83 0.81 1.24 Y63F / C241N 0.76 0.79 1.22 S124A / C241N 0.89 0.78 1.20 Y63F / S124A / C241N 0.60 0.71 1.09

[0064] Example 4: Protein melting temperature (T) of the optimal mutant Y63F of PET hydrolase m Determination of )

[0065] Protein melting temperatures were assessed using differential scanning calorimetry (DSC). The experiment employed a MicroCal PEAQ-DSC differential scanning calorimeter (Malvin Panaco), and the specific method is as follows: the purified protein sample was first subjected to... The protein was concentrated to 1 mg / mL using a 10 kDa ultrafiltration centrifuge (Merck Millipore), and then replaced with reaction buffer (50 mM Gly-OH, pH 9.2) to maintain consistent buffer conditions. 325 μL of the concentrated protein sample and an equal volume of blank reaction buffer were injected into the sample and reference cells, respectively. The temperature program was set to 1.0 °C / min. -1The rate of temperature increase was linear from 25 °C to 95 °C, and the temperature was held constant at 95 °C for 2 minutes to eliminate thermal history effects, and then the rate of temperature decrease was linear from 95 °C to 25 °C. High purity nitrogen (99.999%) was continuously bubbled through the solution during the experiment to prevent evaporation. The results are shown in Figure 1. Figure 1 and Table 3. As can be seen from Table 3, the Tm of the optimal mutant Y63F was increased by 2.5 °C. m

[0066] Table 3. PET hydrolase protein melting temperatures

[0067] Mutant site Protein melting temperature °C HotPETase Y63F ]]> 85.05 Hot PETase 82.50

[0068] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the scope of the present application.​

Claims

1. A PET hydrolase mutant with improved thermal stability, characterized in that, The amino acid sequence of the body variant is SEQ ID NO. 1, which is obtained by any one of the following mutations: Y63F, S124A, S136A, S166A, S169A, N172Q, C241N, T279S, Y63F / S124A, Y63F / C241N, S124A / C241N or Y63F / S124A / C241N.

2. A gene encoding the PET hydrolase mutant with improved thermal stability according to claim 1.

3. The PET hydrolytic enzyme mutant related biomaterial with improved thermal stability according to claim 1, characterized in that, Any one or a combination of the following biological materials: (1) an expression cassette containing the gene according to claim 2; (2) a recombinant expression vector containing the gene according to claim 2; (3) a recombinant expression vector containing the expression cassette according to (1); (4) a recombinant bacterium containing the gene according to claim 2; (5) a recombinant bacterium containing the expression cassette according to (1); (6) a recombinant bacterium containing the recombinant expression vector according to (2) or (3).

4. The biological material according to claim 3, characterized in that: The host bacterium corresponding to the recombinant bacterium in (4), (5) and (6) is selected from prokaryotes, yeasts or higher eukaryotic cells.

5. The biological material according to claim 4, characterized in that: The prokaryote is Escherichia coli, and the yeast is Pichia pastoris.

6. Use of the PET hydrolase mutant with improved thermal stability according to claim 1, the gene according to claim 2 or the biological material according to any one of claims 3-5 in the preparation of a PET hydrolase mutant with improved thermal stability.

7. Use of the PET hydrolytic enzyme mutant of claim 1, the gene of claim 2, or the biological material of any one of claims 3 to 5, characterized in that, One of the following applications: (a) application in the degradation of PET; (b) application in the degradation of plastics.

8. Use according to claim 7, characterized in that: The crystallinity of the PET is 20% or less, further 5-20%, still further 6-8%, and yet further 6.7%.

9. A method of obtaining the PET hydrolytic enzyme mutant of claim 1 with improved thermal stability, characterized in that, The method comprises the following steps: site-directed mutagenesis of a gene encoding a PET hydrolase with an amino acid sequence as shown in SEQ ID NO. 1 to obtain the PET hydrolase mutant with improved thermal stability according to claim 1.

10. The method of claim 9, wherein: The method comprises the following steps: site-directed mutagenesis of a gene encoding a PET hydrolase with an amino acid sequence as shown in SEQ ID NO. 1 to obtain the PET hydrolase mutant with improved thermal stability according to claim 1.

Images